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\begin{document}

\title{Tilletia laevis K\selectlanguage{ngerman}ühn and Tilletia controversa Kühn reprogrammed the
development of wheat anther\selectlanguage{english}}



\author[1]{Li Gao}%
\author[1]{Mingke Fang}%
\author[1]{Delai Chen}%
\author[1]{Ting He}%
\author[1]{Huanyu Jia}%
\author[2]{Yazhen Yang}%
\author[3]{Qingyun Guo}%
\author[1]{Wanquan Chen}%
\affil[1]{Chinese Academy of Agricultural Sciences Institute of Plant Protection}%
\affil[2]{Yangtze University}%
\affil[3]{Qinghai University}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
\emph{Tilletia laevis} cause common bunt and \emph{Tilletia controversa}
cause dwarf bunt of wheat. Both pathogens can cause fungus galls which
replace with wheat grains and lead to the total loss of wheat yields. To
better understand disease progression, confocal microscopy was used to
construct the timeline of cell development of healthy wheat anthers, and
which was approved by analyzing the expression level of some
developmental and program cell death genes. Based on comparing with
normal healthy anthers, tapetum cells should be disappeared at 1300 \selectlanguage{greek}µ\selectlanguage{english}m,
but in \emph{T. laevis} infected anthers, tapetum cells was visible in
2100 \selectlanguage{greek}µ\selectlanguage{english}m of \emph{T. controversa} infected anthers. For the anthers
infected by \emph{T. laevis} and \emph{T. controversa}, cell size and
counts showed significant difference with normal anther cells.
Additionally, for the anthers of five developmental stages,
transcriptome profile and infected five developmental stages were also
analyzed. Furthermore, we established virus-induced gene silencing
(VIGS) for wheat bunt pathogens. Based on RNA-Seq data, \emph{TaEC} in
wheat anther were validated to contribute to the resistance to both bunt
pathogens. This study firstly constructed the development timeline of
wheat anthers and firstly constructed VIGS system to explore the
interaction of both bunt pathogens with wheat anther development.%
\end{abstract}%



\sloppy


\textbf{1 INTRODUCTION}

Wheat is critical to food security worldwide. \emph{Tilletia laevis}K\selectlanguage{ngerman}ühn
lead to common bunt, which seriously reduce wheat grain quality and
yield in wheat growing regions of the world (Albughobeish et al., 2015),
the average yield losses by the bunt pathogens can reach 70-80\% in some
wheat producing areas (Goates et al., 2012). Additionally, wheat dwarf
bunt caused by \emph{Tilletia controversa} Kühn , the teliospores or
bunt sori of the \emph{T. controversa} can last for 10 years in soil and
are easily dispersed from field to field with soil, or can blowout to
new places by infected wheat seeds (Gao et al., 2014). The bunt sori
have a strong odor like of rotting fish, which heavily degrade the
quality of the wheat seeds and flour (Gaudet et al., 2007). Even
slightly low infection can result in clear smell in flour minced from
infected grains (Chen et al., 2016; Nguyen et al., 2019). Also, \emph{T.
controversa} is an internationally important quarantine pathogen and
many countries have strict restriction on importing wheat from areas
where the disease occurs (Mathre et al., 1996; Gao et al., 2014). Both
pathogens grow vigorously and disrupt the normal structure of wheat
anther (JIA et al., 2013; Bokore et al., 2019), which can infect
reproductive organs at different stages of anther development causing
some of the most economically important plant diseases (Ngugi \& Scherm,
2006).

In every anther lobe, cell differentiation and division produce pollen
mother cells (PMC), which are bounded by four somatic layers, epidermis
(EPI), endothecium (EN), middle layer (ML) and tapetum (TA) from the
outside to inside (Scott et al., 2004; Chang et al., 2011; Xu et al.,
2016; Browne et al., 2018). EPI prevent water loss and have role in
dehiscence and in gas exchange, EN and ML have a role in cell structure
and support to the other cells (Goldberg et al., 1993; Sun et al.,
2007). TA plays vital roles in supporting pollen grain development
succeeding the completion of meiosis and meiotic cytokinesis (Zhang et
al., 2006; Browne et al., 2018). Additionally, TA is the innermost layer
of the anthers and have a role to provides the nutrients to the emerging
pollen grain and it undergoes to program cell death in the later stages
of pollen grain development. Microspores (MP) are involved in the pollen
grain and sperm cell development (Zhang \& Wilsonc, 2009). Anther is
particularly more sensitive to biotic and abiotic factors (Hedhlyet al.,
2009) during development (Saini \& Westgate, 1999; Dolferus et al.,
2011). \emph{Ustilago maydis} causes disturbance in the anther lobes and
infected organs (Walbot \& Skibbe, 2010) which changed the cells of
anther lobes of maize i.e. EPI, EN, SPL, ML and TA, which are involved
in the development of pollen mother cells (Gao et al.,
2013).\emph{RAFTIN} have a role in TA development of cereal crops (Wang
et al., 2003). \emph{MYB} transcription factors have a role in anther
development in barley and other cereal crops (Murray et al.,
2003).\emph{TaMYB80} influence the pollen grain development in
participating the program cell death of TA cells (Gao et al., 2013;
Millar \& Gubler, 2005; Xue, 2005)

High-throughput gene expression analysis using RNA-Seq signifies a very
powerful tool for transcriptomic description of plants during the
interaction of pathogens and plants (Wang et al., 2009; Massart et al.,
2014). The RNA-Seq has been performed in many crops against different
pathogens, such as banana and \emph{Fusarium oxysporum} f.
sp.\emph{cubense} (Li, 2013), mango and \emph{Fusarium mangiferae} (Liu
et al., 2016), potato and \emph{Phytophthora infestants} (Gao et al.,
2013) and so on.

Virus-induced gene silencing (VIGS) has been developed as an effective
genetics tool for assessing gene functions in different plants species
including maize, pea, wheat, tobacco, tomato and barley (Constantin et
al., 2004; Scofield et al., 2005; Ding et al., 2006). Previous studies
revealed functional analysis of wheat genes against \emph{Zymoseptoria
tritici} resistance and susceptibility (Lee et al., 2015) used
for\emph{Lr21} -mediated leaf rust resistance pathway in hexaploid wheat
(Gao et al., 2013), and established in wheat spikes and grains for
HMW-GS-encoding genes, in which clear photobleaching symptoms were
appeared in silenced plants (Ma et al., 2012).

In this study, we used confocal microscopy to build up the timeline of
wheat anther development, which showed the characteristics of cell types
in wheat anther, and the anther timeline was approved by section and
quantification of anther developmental genes using qRT-PCR assays. In
addition, RNA-Seq was used to analyze wheat anther stages transcriptome
changes in response to \emph{T. laevis} and \emph{T.
controversa}infection. VIGS was used to silence one of anther
development genes in RNA-Seq to explore the function of anther genes in
the infection process of \emph{T. laevis} and \emph{T. controversa} .
This is the first report of the bunt pathogens reprogrammed the anther
development based on confocal microscope and RNA-Seq analysis, and
explore related gene's function by VIGS technique.

\textbf{2 MATERIALS AND METHODS}

\textbf{2.1 Plant and fungal material}

Highly susceptible wheat cultivar Dongxuan 3 was obtained from Institute
of Plant Protection, Chinese Academy of Agricultural Sciences,
China.\emph{T. laevis} isolate was isolated from Henan province, China
and\emph{T. controversa} was gifted by Blair Goates, the United States
Department of Agriculture (USDA), Agricultural Research Service (ARS),
USA. \emph{T. laevis} was propagated in water agar medium (20 g/liter
agar powder), while \emph{T. controversa} was propagated in soil agar
medium (20 g agar powder, 75 g soil in a liter distilled water) and
autoclaved for pouring the media into sterilized plates. \emph{T.
laevis} plates were incubated at 16 °C in a 24 h light in incubator (MLR
352H, Panasonic, USA) after covering with parafilm for 7 days
incubation. Mycelium production were observed under an automated invert
fluorescence microscope (IX83, Olympus, Japan). Hyphae were collected
with distilled water and used to inoculate wheat plants. The same method
of incubation was adopted for \emph{T. controversa} except the cultured
plates were kept at 5 °C for 60 days.

\textbf{2.2 Inoculation of \emph{T. controversa} and \emph{T. laevis}}

For inoculation, wheat seeds were sterilized with 30\% sodium
hypochlorite for 5 min, rinsed 5 times in water, and germinated for 30
days in incubator (AUCMA, Qing Dao, China) to vernalize. Then seedlings
were grown in a 1:2 mixture of organic matter (peat moss, Beijing,
China) and soil (Beijing, China) in pots. Seedlings for the \emph{T.
controversa} experiment were grown in a 14 h light/10 h dark at 5 °C
while for the \emph{T. laevis} experiment at 15 °C to the tillering
stage and both at 25 °C at the boot stage. At the early boot stage, the
spikes were injected with 1 ml inoculum suspensions of \emph{T.
controversa} or \emph{T. laevis} when the young tassels of wheat were
still wrapped by leaf sheaths. The suspension contained infectious
hyphae at the concentration of 10\textsuperscript{6} cfu/ml with the
OD\textsubscript{600} of 0.15. Inoculation was repeated 5 times with one
day interval. Plants injected with sterilized ddH\textsubscript{2}O were
grown under the same conditions for use as the control treatment.

\textbf{2.3 Molecular detection of \emph{T. laevis} and \emph{T.
controversa} in inoculated wheat anther}

DNA was extracted from wheat leaves using the cetyl trimethylammonium
bromide (CTAB) method. The sequence characterized amplified region
(SCAR) markers for \emph{T. laevis} (Yao et al., 2019) and \emph{T.
controversa} (Gao et al., 2014) were used as previously described.
Successful infection was indicated by positive amplification of the
660-bp (\emph{T. laevis} ) or 372-bp (\emph{T. controversa} ) specific
band.

\textbf{2.4 Anther staining and confocal microscopy}

The spikelets were dissected from the spike, and anthers were removed
from each spikelet under a stereo microscope (S6D, Leica, Germany).
Depending on the experiment situation, anthers dipped in absolute
ethanol for 30 min. Application of absolute ethanol was repeated thrice
after 30 min until tissue change into white color. Hyphae in anthers
were stained with chitin specific dye WGA-AF 488 (Invitrogen, Eugene,
USA) and anther cells were stained with propidium iodide (PI)
(Invitrogen, Eugene, USA). Samples were incubated at
25\textsuperscript{o}C for 60 min in 0.02\% Tween 20 containing
WGA-AF488 and PI at the ratio of 1:2. After staining, samples were
rinsed 4 to 6 times in 1× PBS (pH 7.4) (Suolaibao, Beijing, China) and
stored in PBS at 4 °C without light. The fungal staining was performed
at 25 °C. Samples were observed under a confocal laser scanning
microscope (Leica SP8, Germany) with excitation 488 nm/emission 510-550
nm (for WGA-AF 488) and excitation 561 nm/emission 570-730 nm (for PI).

\textbf{2.5 RNA extraction and quantitative Real-time PCR (qRT-PCR)}

RNA from anthers was extracted using the total RNA pure kit (Aidlab
Biotech, Beijing, China) as per the manufacturer's protocol. The
concentration of RNA in each sample was determined using a NanoDrop
(Denovix, Spectrophotometer, USA). RNA was stored at -80 \selectlanguage{english}@C until used.
Aerosol protected pipette tips were used throughout the extraction and
quantification steps to prevent RNA contamination. The first-strand cDNA
was synthesized by using 2000 ng \selectlanguage{greek}µ\selectlanguage{english}l\textsuperscript{-1} purified total
RNA, RT/RI enzyme and oligo (dT)\textsubscript{18} primer (TransGen
Biotech, Beijing, China) following the instructions of kit. The qRT-PCR
was done using Top Green qPCR SuperMix in a volume of 20 \selectlanguage{greek}µ\selectlanguage{english}l according to
the manufacturer's instructions and applied to the QuantStudio 5
real-time PCR system (Applied Biosystems, Beijing, China). The
amplification of the wheat actin was used as an internal control for
normalizing all data.\emph{Actin} gene were used as a negative control
in this experiment. All genes were run on a 96-well QuantStudio 5
(Applied Biosystems, Beijing, China) with the following conditions:
pre-denaturation at 95 \selectlanguage{ngerman}°C for 10 min, 40 cycles (95 °C for 15 s, 58 °C
for 30 s and 72 °C for 30 s) were performed. The 2
\selectlanguage{english}\textsuperscript{-ΔΔCt} method (Pfaffl, 2001) was used to evaluate the
presence of genes with three biological replicates and four technical
replicates, which prove the timeline of anther development.

\textbf{2.6 Total RNA extraction and library preparation for wheat
anthers of \emph{T. laevis} and \emph{T. controversa}}

Anthers were collected from 5 different growth periods
(0\textasciitilde{}150 \selectlanguage{greek}μ\selectlanguage{english}m, 150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m,
400\textasciitilde{}700 \selectlanguage{greek}μ\selectlanguage{english}m, 700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m and
1100\textasciitilde{}1300 \selectlanguage{greek}μ\selectlanguage{english}m) frozen in liquid nitrogen and stored at
-80 @\selectlanguage{english}C for later use. RN53-EASYspin Plus Complex Plant RNA Kit (Adlai,
Beijing-China) was used to extract total RNA according to the
manufacture's instruction. The quality of the purified RNA was measured
on the 260/280 nm and 260/230 nm absorbance ratio obtained, using a
NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA,
USA) and 1\% agarose gel electrophoresis was used to detect RNA
integrity. RNA-seq transcriptome library was prepared by following
TrueSeq RNA sample preparation kit from Illumina (San, Deigo, CA) using
1 \selectlanguage{greek}µ\selectlanguage{english}g of total RNA. The first cDNA synthesized using random primer
(oligonucleotides) (Illumina, San Diego, CA, USA). Second-cDNA strand
synthesized by DNA polymerase I, RNase H, dNTPs and buffer solution.
cDNA fragments were purified using QIAquick PCR kit (Qiagen, Venlo,
Netherland). These cDNA fragments washed by EB buffer for addition of
end reparation Poly (A) and then ligated with special Illumina
sequencing adapters. The final cDNA library was constructed by
purification of the cDNA small fragments, which were enriched by PCR
products. Sequencing was done by using Illumina HiSeq 2500 platform
(Illumina, USA) by Shanghai Meiji Biomedical Technology Co., Ltd.

\textbf{2.7 Quality control and read mapping}

The raw paired end reads were trimmed and quality controlled by SeqPrep
(\emph{https://github.com/jstjohn/SeqPrep}) and sickle
(\emph{https://github.com/najoshi/sickle}) with default parameters. The
parameters were as follows: 1) trimming the 3'-end low-quality bases
(quality value less than 20); 2) removing N-containing reads, 3)
discarding adaptor; 4) sequence with length < \selectlanguage{english}30 bp after quality
trimming. Finally, the clean reads were mapped to reference genome using
Bowtie2 (version 2.2.9) software. The mapped reads of each sample were
assembled by StringTie
(\emph{https://ccb.jhu.edu/software/stringtie/index.shtml?t=example}) in
a reference-based approach (Pertea et al., 2015).

\textbf{2.8 Differentially expression analysis and functional
enrichment}

The clean reads of each sample were assembled by using StringTie
software. FPKM (fragments per kilobase of transcript per million mapped
reads) value of every transcript was analyzed and calculated by using
StringTie software. Additionally, the DGEs (differentially expressed
genes) were recognized by using the technique DESeq software in
both\emph{T. laevis} and \emph{T. controversa} infected and control
samples. The genes/transcripts with the parameter of false discovery
rate (FDR) < \selectlanguage{english}0.05 and absolute fold change [?] 2 were considered
differentially expressed genes/transcripts (Samac et al., 2011). In
addition, functional-enrichment analysis including GO and KEGG were
performed to identify which DEGs by using R based on the hypergeometric
distribution test with the FDR < \selectlanguage{english}0.05 represents the significantly
expressed transcripts (Kanehisa et al., 2008).

\textbf{2.9 Validation of transcript data}

The first-strand cDNA was synthesized by using 1.5 \selectlanguage{greek}µ\selectlanguage{english}g purified RNA by
using Trans Script One-Step gDNA Removal and cDNA Synthesis SuperMix Kit
(TransGen Biotech, Beijing, China) and stored at -20 @\selectlanguage{english}C for further use.
PerfectStart Green qPCR SuperMix (TransGen Biotech, Beijing, China) kit
was used for qRT-PCR reactions. For validation of transcripts data of
significant DEGs, 11 genes were selected from both pathogen infected and
control samples for validation analysis.

\textbf{2.10 Establishment of virus-induced gene silencing (VIGS) to
wheat bunt pathogens}

The cDNA fragment of \emph{TaEC} with \emph{ApaI} restriction site were
obtained by reverse transcription PCR and inserted into the original
Barley strip mosaic virus (BSMV) vector for gene silencing. Fragment had
no similarity to any other wheat gene in BLAST analysis (Http://
blast.ncbi.nlm.nih.gov/Blast/). In addition, to confirm the specificity
of fragments used in the VIGS assays, the expression level of\emph{TaEC}
were measured in \emph{TaEC-} knockdown plants, respectively. The
fragment of wheat phytoene desaturase (TaPDS) was inserted into the BSMV
vector as the positive control.

The BSMV viruses (BSMV:\selectlanguage{greek}γ, \selectlanguage{english}BSMV:TaPDS; BSMV: TaEC) were inoculated in
wheat tassel at (0, 10, 15, 22, 35 and 42d) and then inoculated \emph{T.
controversa} and \emph{T. laevis} at 5 dpi following modification of
previous method. Virus infection on \emph{Nicotiana benthamiana} were
achieved by \emph{Agrobacterium} -mediated transient gene expression of
infectious constructs from the T-DNA of a binary plasmid (GV3101). The
bacterial solution was centrifuged at 5000rmp for 10 min, resuspended in
infiltration buffer (10 mM MgCl\textsubscript{2}, 10 mM 2-(N-morpholino)
ethanesulfonic acid (MES) and 0.1 mM acetosyringone (AS) and adjusted to
0.7 (OD\textsubscript{600})and then incubated at room temperature for 2
h. The culture was then infiltrated to the underside of a leaf using a 1
ml syringe without a needle. The \emph{N. benthamiana} leaves were
grounded in the pestle and mortar and were inoculated on the wheat
leaves at the booting stage and repeated three times. At the same time,
the wheat inoculated with BSMV\selectlanguage{greek}γ \selectlanguage{english}was used as the control. The disease
index was analyzed between BSMV\selectlanguage{greek}γ \selectlanguage{english}and BSMV: TaEC. RNA was extracted from
the ears of the infected plants and the control group using the plant
total RNA extraction kit (Tiangen, Beijing). The integrity,
concentration and purity of RNA were detected by electrophoresis of
agarose gel and Spectrophotometer (DeNovix DS-11+Spectrophotometer,US).
According to \emph{TaEC} sequences using the SnapGene design primers
(AGGGACAACCATTTCCAAGGATGCC/CGCGTTCTGGCAA

GTACTCTCG), wheat \emph{Actin} gene was used as the internal reference
gene for qRT-PCR detection of transcripts. SuperReal PreMix Plus(SYBR
Green) kit (Tiangen, Beijing) was used to perform qRT-PCR reaction, the
20 \selectlanguage{greek}µ\selectlanguage{english}l reaction mixtures contain 1 \selectlanguage{greek}µ\selectlanguage{english}l template, 0.4 of each primer (10
\selectlanguage{greek}μ\selectlanguage{english}M), 10 \selectlanguage{greek}µ\selectlanguage{english}l SuperMix (TransGen Biotech, Beijing, China) and 8.2 \selectlanguage{greek}µ\selectlanguage{english}l
ddH\textsubscript{2}O. The amplification protocol was 95  for 15 min,
followed by 40 cycles of 95  for 10 s, 60  for 32s. All cDNA and
qRT-PCR reactions were carried out in triplicate. The
2\textsuperscript{-[?][?]CT} method was used to calculate the relative
expression of each gene (Ma et al., 2012).

\textbf{2.11 Statistical analysis}

Data analysis was performed using Microsoft excel 2016 and Volocity
version 6.0 (www.perkinelmer.com.cn) image processing software.
Student's \emph{t-test} *\emph{P} [?] 0.05, ** \emph{P} [?] 0.01 with three
replications were applied to calculate the cell counts and cell size.
Values represent the average standard error of three independent
biological replicate.

\textbf{3 RESULTS}

\textbf{3.1 Establishment of anther length with timeline and cell type
specification in the anther}

Anther development from primordium within developing spikelets on a
spike to a pollen shed, corresponding to anther lengths of 100 to 2300
\selectlanguage{greek}µ\selectlanguage{english}m (or more depending on the cultivar), respectively. Subsequently,
anther length is gradual and goes to stabilization. To correlate length
and time, anthers were dissected from spikelets of spike and the
neighboring spikelets were dissected minimum 24 h later from the same
position. In Figure \textbf{1} , results showed that anthers got double
in length from 100 to 200 \selectlanguage{greek}µ\selectlanguage{english}m in 2.5 days in the passage from day 1 to
3.5, and double again to 400 by day 7, and then the elongation in length
decrease gradually over the 32 days to achieve 2300 \selectlanguage{greek}µ\selectlanguage{english}m. The floral
meristem consists of three germ layers: namely L1, L2, and L3, which are
involved to make pollen grains finally in the last stage. Except the
EPI, vascular system (VS) and connective tissue (CT), which are the
result of division of L1 and L3 cells, respectively, while L2 form AR
cells from the four corners of each anther primordium. The mitotic
division of the AR cells occur which produce EN, SPL and PMC cells. The
SPL then divide to form ML and TA cells. Subsequently, PMC undergoes
meiosis to form Tds, which differentiate into MP rapidly and MP
differentiate into PG after cell division. The AR cells appear in
\textless{} 150 \selectlanguage{greek}µ\selectlanguage{english}m anthers and then disappear at \textgreater{} 150 \selectlanguage{greek}µ\selectlanguage{english}m
after formation of EN, SPL and PMC cells. Subsequently, SPL cells
disappear almost at 400 \selectlanguage{greek}µ\selectlanguage{english}m after formation of ML and TA cells.
Similarly, PMC cells disappear near to 700 \selectlanguage{greek}µ\selectlanguage{english}m after the formation of
microspore (MP) which differentiate into pollen grain (PG) which
released in the environment by opening the epidermal cells from stomium
region.

\textbf{3.2 The tissue appearance in normal anthers under confocal
microscopy}

Timeline was used to differentiate the locule of anthers spanning 32-day
period from primordium to pollen grain. The appearance and division of
locular cells were gritty, and key actions were listed in
Figure\textbf{2} and shown morphology of important cell types in
confocal images \textbf{(} Figure \textbf{2A-I).} At least 60 anthers of
each cell type were analyzed. The anthers smaller than 100 \selectlanguage{greek}µ\selectlanguage{english}m, no AR
cells were seen; instead L2 layer in every locule was present arranged
in a disorganized manner. After AR cells specification, ready for
mitosis to produce the somatic (EN and SPL) and reproductive (PMC)
cells. In 100 \selectlanguage{greek}µ\selectlanguage{english}m anthers, AR cells were clearly visible and present in
the centre of locule (Figure \textbf{2A} ). After the formation of AR
cells, the somatic tissues become ready to pattern. According to our
results AR cells differentiate to EN, SPL and PMC cells in 310 \selectlanguage{greek}µ\selectlanguage{english}m long
anthers (Figure \textbf{2B} ). According to linkage model, EN cells
differentiate with SPL, which differentiate into ML and TA cells.
Indeed, in 310 to 580 \selectlanguage{greek}µ\selectlanguage{english}m anthers, the four cells were differentiated
clearly, which are named as the EPI, SPL, EN and PMC cells (Figure
\textbf{2B-E} ). At 580 \selectlanguage{greek}µ\selectlanguage{english}m, EN cells remain oval, while at 380 \selectlanguage{greek}µ\selectlanguage{english}m SPL
showed round, and at 370 \selectlanguage{greek}µ\selectlanguage{english}m PMC cells has round like structures,
indicative that these three have developed different identities (Figure
\textbf{2C-E)} . PMC cells undergoes meiosis and produce the tetrads
cells (Tds) (Figure\textbf{2F-G} ). Additionally, the Tds cells
generates the young MP cells (Figure \textbf{2H} ). The MP raise larger
and more spherical, and changed into PG. In the last stage, PG filled
with the starch and released out by opening the stomium (Figure
\textbf{2I} ).

\textbf{3.3 Approval of anther timeline based on specific genes
quantification}

We approved the timeline by analyzing the TA developmental and TA
program cell death genes by using in qRT-PCR. We divided the anthers
into five range i.e. 200-300 \selectlanguage{greek}µ\selectlanguage{english}m, 400-600 \selectlanguage{greek}µ\selectlanguage{english}m, 700-900 \selectlanguage{greek}µ\selectlanguage{english}m, 1100-1300 \selectlanguage{greek}µ\selectlanguage{english}m
and 1400-2300 \selectlanguage{greek}µ\selectlanguage{english}m. \emph{RAFTIN1} and \emph{TaMYB80} have a role in TA
development and TA program cell death, respectively. According to the
timeline, TA appear in 400 \selectlanguage{greek}µ\selectlanguage{english}m long anthers and disappear in 1300 \selectlanguage{greek}µ\selectlanguage{english}m
anthers (Figure \textbf{1} ). Relative expression of both genes in
200-300 \selectlanguage{greek}µ\selectlanguage{english}m anthers can show the presence of genes. The expression
of\emph{RAFTIN1} reached at its peak in 400-600 \selectlanguage{greek}µ\selectlanguage{english}m anthers
while\emph{TaMYB80} showed little expression. This observation showed
that TA development has been start in this size of anthers. Similar
response was observed in 700-900 \selectlanguage{greek}µ\selectlanguage{english}m anthers. On the other hand, the
highest expression of \emph{TaMYB80} was noted in 1100-1300 \selectlanguage{greek}µ\selectlanguage{english}m anthers
and vice versa in \emph{RAFTIN1} . This is because of that TA started to
degrade after 1100 \selectlanguage{greek}µ\selectlanguage{english}m anthers and disappear in 1300 \selectlanguage{greek}µ\selectlanguage{english}m anthers. In the
case of 1400-2300 \selectlanguage{greek}µ\selectlanguage{english}m anthers, the expression of both genes not enough to
prove the presence of TA in the anther length. Such expression level
therefore provides information to approve the timeline \textbf{(} Figure

\textbf{S1).}

\textbf{3.4 Cell-type-specific programs of division and expansion}

The final form of an organ reflects the pattern of cell proliferation,
expansion, and differentiation of the constituent tissues and their cell
type. For every cell type, cell counts along X-, and Y- axes
(Figure\textbf{3AB} ) and cell width, cell length and cell depth were
quantified in mm along X-, Y-, and Z-axes, respectively (Figure
\textbf{3E-G} ) at least in 12 anthers per stage with > \selectlanguage{english}10 replicates.
We analyzed the cell ratio of Y-axis and X-axis (Figure \textbf{3I} ).
These values were multiplied to estimate the cell size and cell counts
per anther, resolved by the tissue type (Figure \textbf{3CH} ).
Additionally, percentage in every anther locule was calculated as well
(Figure\textbf{3J} ). As shown in Figure \textbf{3A,} Table \textbf{1} ,
EPI cells have consistently more cells counts along X-axis then other
cell types and their ratio increased more rapidly from 200-400 \selectlanguage{greek}µ\selectlanguage{english}m
anthers moves towards the stability until to maturation, where pollen
grains released. The cell counts of all other cell types increased
gradually as anther undergoes development. In Figure \textbf{3B,} Table
\textbf{1,}EN cells has greater cell counts then all other type of cells
along Y-axis. Results showed that the cell counts of EN cells along
Y-axis increased extensively during the anther development from 400-800
\selectlanguage{greek}µ\selectlanguage{english}m compared to other length of development. EPI increased more rapidly
from 200-400 \selectlanguage{greek}µ\selectlanguage{english}m and then become stable. As shown in Figure \textbf{3C,}
Table\textbf{1} , the total cell/locule (X*Y) of EN cells was more than
all other cell types during anther development, except from 200-700 \selectlanguage{greek}µ\selectlanguage{english}m,
where cell counts of EPI was more. Moreover, total cell/locule of SPL,
ML, TA, PMC, MP and PG increased as the anther increased in size.
Interestingly, the percentage of locule cell count of EPI cells
decreased during development, but increased in EN cells with the anther
development. However, almost irregular response was noted in SPL, ML,
TA, PMC, MP and PG (Figure \textbf{3D,} Table \textbf{1} ). As shown in
Figure \textbf{3E,} Table \textbf{2,} width (X-axis) of all cell types
increased as anther goes to development. The maximum cell width noted in
EN cells, but interestingly PG got more width suddenly in anthers
2000-2300 \selectlanguage{greek}µ\selectlanguage{english}m than other cell types. In addition, width of SPL cells only
recorded in 200-400 \selectlanguage{greek}µ\selectlanguage{english}m anthers and then disappear. The irregular pattern
of cell width of all cell types was recorded in EPI, ML, TA and PG
during cell development. The maximum cell length (Y-axis) was recorded
in EPI cells compared to all other cell types. However, in EN, ML, TA,
PMC and MP length increased gradually during anther development. PG
developed more rapidly especially from 1700-2000 \selectlanguage{greek}µ\selectlanguage{english}m. Additionally, the
length of SPL cells increased slowly, then cells become disappear by
differentiating into ML and TA after division (Figure \textbf{3F,}
Table\textbf{2} ). Likewise, width and length, the cell depth (Z-axis)
of all cell types significantly influenced during the division and
expansion. Results demonstrated that cell depth of EPI, EN, SPL, ML, TA,
PMC and PG increased gradually as anther increased in size during
development, but in PG depth increased more rapidly from 1700-2000 \selectlanguage{greek}µ\selectlanguage{english}m
long anthers and then moves towards stability (Figure \textbf{3G,} Table
\textbf{2} ). The cell volume (X*Y*Z) was calculated for all cell types
sizes and more cell volume was observed in the PG compared to others
cell types. While in ML and TA suppressed proliferation of cells were
noted (Figure\textbf{3H,} Table \textbf{2} ). Length/ width (Y/X) ratio
was calculated for all cell types. EPI cells showed more percentage
during their expansion and division, while all other cells showed
gradual response during the division (Figure \textbf{3I,} Table 2).
Percentage of locule volumes were calculated as well and PG has maximum
in later stage of development (Figure \textbf{3J,} Table \textbf{2} ).

\textbf{3.5 Fungal detection in infected anthers}

We extracted DNA from \emph{T. laevis} and \emph{T. controversa}infected
anthers to detect the fungus\emph{.} The expected 660 bp band of
\emph{T. laevis} was detected in the infected anther samples
(Figure\textbf{S2a} ) and expected 372 bp band was detected from the
\emph{T. controversa} infected anther samples (Figure \textbf{S2b} ).
Based on the detection of the specific SCAR of both pathogens, we
recognized the tested anthers were infected successfully by \emph{T.
laevis} or\emph{T. controversa} .

\textbf{3.6 Effects of \emph{T. laevis} infections on wheat anther
development}

We studied the effects of \emph{T. laevis} on the development of EPI,
EN, TA and PG cells in wheat anthers. Our results showed that the hyphae
were seen in EPI cells at 800 \selectlanguage{greek}µ\selectlanguage{english}m (Figure \textbf{4A-C} ). As shown in
Figure \textbf{4D-F} , the fungus was growing from EPI to EN cells when
anthers were growing from 800 \selectlanguage{greek}µ\selectlanguage{english}m to 1220 \selectlanguage{greek}µ\selectlanguage{english}m. Hyphae were also seen in TA
and PG cells when anther reached 1300 \selectlanguage{greek}µ\selectlanguage{english}m and 1520 \selectlanguage{greek}µ\selectlanguage{english}m, respectively
(Figure \textbf{4G-L} ). All above results suggested that fungus grows
from EPI cells to PG as anther grow. Additionally, according to
timeline, normal TA cells should disappear at 1300 \selectlanguage{greek}µ\selectlanguage{english}m (Figure\textbf{1}
), but TA was still present in 1300 \selectlanguage{greek}µ\selectlanguage{english}m in \emph{T. laevis}infected
anthers. These changes showed irregular development of anther cells
after fungal infection.

\textbf{3.7 Cell differentiation and expansion in \emph{T.
laevis}infected anthers}

Cell numbers (Figure\textbf{5A-D} ) and sizes (Figure \textbf{5E-G} ),
cell volume (Figure\textbf{5H} ) and shapes (y/x) (Figure \textbf{5I} )
in the \emph{T. laevis} infected anthers were showed. Specifically, In
Figure\textbf{5A,} Table \textbf{3} , after \emph{T. laevis} infection,
cell counts of EPI, EN, SPL, ML, TA, PMC, MP and pollen grain (PG) along
X-axis changed during anther development, and significantly increased
response was noted in the ML of 700-1100 \selectlanguage{greek}µ\selectlanguage{english}m anthers. For EN, cell counts
were increased in 250-400 \selectlanguage{greek}µ\selectlanguage{english}m, and almost keep the same between 700-2300
\selectlanguage{greek}µ\selectlanguage{english}m. Additionally, ML cells had increased cell counts during 400-800 \selectlanguage{greek}µ\selectlanguage{english}m
anthers \textbf{(} Figure \textbf{5B,} Table \textbf{3).} The total
cells per locule (X*Y) of EPI cells increase from 200-2300 \selectlanguage{greek}µ\selectlanguage{english}m, while in
EN cells increased much more in 200-400 \selectlanguage{greek}µ\selectlanguage{english}m anthers. In the case of ML
and TA, total cells per locule increased in 400-1100 \selectlanguage{greek}µ\selectlanguage{english}m and 700-1200 \selectlanguage{greek}µ\selectlanguage{english}m
respectively (Figure \textbf{5C,} Table \textbf{3)} . The percentage of
locule cell count of EPI cells statistically increased from 200-700 \selectlanguage{greek}µ\selectlanguage{english}m
infection. In contrast, the percent of EN cells significantly decreased
in 200-700 \selectlanguage{greek}µ\selectlanguage{english}m anther. However, the percentage of locule cell count of MP
and PG almost keep the same \textbf{(} Figure \textbf{5D,}
Table\textbf{3)} . Results demonstrate that there was no significant
change in the cell width (X-axis) of TA, while for EN, which increased
from200-2300 \selectlanguage{greek}µ\selectlanguage{english}m anthers (Figure \textbf{5E,} Table \textbf{4).} The cell
length (Y-axis) significantly increased only in EPI cells during anther
development in 1100-1300 \selectlanguage{greek}µ\selectlanguage{english}m anthers (Figure \textbf{5F,} Table\textbf{4}
). For cell depth (Z-axis), the infected anthers did not show much
change in EPI, EN, SPL, ML, TA, PMC, MP cells in the developmental
period (Figure \textbf{5G,} Table \textbf{4} ). The volume (X*Y*Z) of
EPI and EN cells increased from 150-2300 \selectlanguage{greek}µ\selectlanguage{english}m anthers. (Figure
\textbf{5H,}Table \textbf{4} ). Likewise, Y/X of EN cells decreased
during early anther development. The Y/X of SPL, ML, MP, PG cells almost
keep the same during anther development (Figure \textbf{5I,} Table
\textbf{4} ). The percentage of locule volume of EPI, EN cells
significantly decreased in 150-400 \selectlanguage{greek}µ\selectlanguage{english}m and then increase (Figure
\textbf{5J,} Table \textbf{4)} . All above changes in the anther cell
types suggested that \emph{T. laevis} infects the anther structure and
create morphological changes.

\textbf{3.8 Effects of \emph{T. controversa} infections on wheat anther
development}

The effects of \emph{T. controversa} on the development of EPI, EN, TA
and PG cells in wheat anthers were also analyzed. We found hyphae in EPI
cells of anther at 1000 \selectlanguage{greek}µ\selectlanguage{english}m (Figure \textbf{6A-C)} . Hyphae were also
seen in TA and PG cells with 2100 \selectlanguage{greek}µ\selectlanguage{english}m anther a (Figure \textbf{6G-L} ).
Additionally, according to timeline, TA cells should disappear at 1300
\selectlanguage{greek}µ\selectlanguage{english}m (Figure \textbf{1} ), but TA was present in 2100 \selectlanguage{greek}µ\selectlanguage{english}m of \emph{T.
controversa} infected anthers.

\textbf{3.9 Cell differentiation and expansion in \emph{T.
controversa}infected anthers}

Cell numbers (Figure \textbf{7A-D} ), sizes (Figure \textbf{7E-G} ),
volume \textbf{(} Figure \textbf{7H} ) and shapes (y/x)
(Figure\textbf{7I} ) of the \emph{T. controversa} infected anthers were
showed. The cell count of EPI, EN cells along X-axis were increased in
developmental anthers. However, SPL, TA, PMC, MP and PG had no
statistically difference along the X-axis after \emph{T.
controversa}infection (Figure \textbf{7A,} Table \textbf{5)} . The cell
count of EPI and EN cells along Y-axis increased during 150-800 and then
keep the number until2300 \selectlanguage{greek}µ\selectlanguage{english}m. MP and PG cells almost keep the same
during development (Figure \textbf{7B,} Table \textbf{5} ). The total
cell/locule (X*Y) of EPI and EN cells increased in 150-2300 \selectlanguage{greek}µ\selectlanguage{english}m
after\emph{T. controversa} infection. Similarly, TA increased from
400-1300 \selectlanguage{greek}µ\selectlanguage{english}m (Figure \textbf{7C,} Table \textbf{5} ). EPI and EN cells
showed reverse tend in the development anthers (Figure \textbf{7D,}
Table\textbf{5} ). Results showed that the cell width (X-axis) of EPI,
EN, SPL, PG increased during anther development, respectively\textbf{(}
Figure \textbf{7E,} Table \textbf{6)} . Increased cell length (Y-axis)
of EPI cells were recorded during anther development. EN and PMC cells
did not showed much change during development (Figure\textbf{7F,} Table
\textbf{6)} . Cell depth (Z-axis) of EPI, and TA cells did not show much
change (Figure \textbf{7G,} Table \textbf{6} ). The volume (X*Y*Z) of
EPI, EN, TA and PMC cells did not show much more changes (Figure
\textbf{7H,} Table \textbf{6)} . The Y/X ratio of EN and PG cells not
change too much, while EPI cells showed decrease tend during anther
development (Figure \textbf{7I,} Table \textbf{6)} . The percentage of
locule volume of EPI, EN and TA cells indicated similar changes.
Additionally, percentage of PMC, MP and PG showed increased in anther
development (Figure \textbf{7J,} Table \textbf{6} ).

\textbf{3.10 Transcriptome analysis after \emph{T. laevis} and \emph{T.
controversa} infection}

In order to known the transcriptional changes in wheat anther
developmental stages after \emph{T. laevis} and \emph{T.
controversa}infection, we analyzed anthers of different lengths
including; 0\textasciitilde{}150 \selectlanguage{greek}μ\selectlanguage{english}m, 150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m,
400\textasciitilde{}700 \selectlanguage{greek}μ\selectlanguage{english}m, 700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m and
1100\textasciitilde{}1300 \selectlanguage{greek}μ\selectlanguage{english}m by RNA-Seq. The raw reads were obtained by
removing the adaptor sequence, ambiguous bases, and low-quality
sequences. For \emph{T. laevis} , approximately, 70.74 (control-2-3) to
91.88 (control-2-2) million clean reads were obtained from control
samples and their mapping rate were 85.76\% to 69.10\%, respectively,
and clean reads in \emph{T. laevis} infected samples were 75
(infected-5-2) to 108.29 (infected-2-3) million reads and their mapping
rate were 68.57\% to 83.39\%, respectively. The Q20, Q30, GC content and
mapped reads of above mentioned transcripts ranged from 98.51 to 98.76
\%, 95.56 to 96.17\%, 52.89 to 54.98\% and 51.94 to 90.30 million,
respectively (Table \textbf{1} ). For \emph{T. controversa} ,
approximately 89.77 (control-2-3) to 104.68 (control-2-2) million clean
reads were obtained from control samples and their mapping rate were
87.26 \% to 87.04\%, respectively, and clean reads in \emph{T.
controversa} infected samples were 88.30 (infected-1-1) to 121.96
(infected-5-3) million reads and their mapping rate were 82.62 \% to
83.71\%, respectively. The Q20, Q30, GC content and mapped reads of
above mentioned transcripts ranged from 98.24 to 98.52\%, 94.78 to
95.46\%, 53.16 to 54.77 \% and 74.33 to 92.40 million, respectively
(Table \textbf{S2} ). In order to clarify the reproducibility between
samples, we analyzed and calculated the individual correlation
coefficients between samples through principal component analysis (PCA).
The biological replicates of each treatment were clustered together to
prove that there was good repetition between replicates and there were
differences between treatments for \emph{T. controversa}
(Figure\textbf{S3A} ) and for \emph{T. laevis} (Figure \textbf{S3B} ).

\textbf{3.11 Identification and analysis of differentially expressed
genes after \emph{T. controversa} and \emph{T. laevis} infection}

Gene expression comparison were performed among \emph{T. controversa}
,\emph{T. laevis} and their control samples at five different anther
stages. For \emph{T. controversa} , at \textless{}150\selectlanguage{greek}μ\selectlanguage{english}m, 3293 DEGs were
identified, of which 2644 were up-regulated and 649 were down-regulated.
For 150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m anther length libraries, 2916 DEGs were
identified, of which 1837 were up-regulated and 1079 were
down-regulated. Similarly, for 400\textasciitilde{}700 \selectlanguage{greek}μ\selectlanguage{english}m, 3730 DEGs
were generated, of which 1126 were up-regulated and 2604 were
down-regulated. For 700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m, 1118 DEGs were
identified, of which 767 were up-regulated and 351 were down-regulated.
While for 1100\textasciitilde{}1300 \selectlanguage{greek}μ\selectlanguage{english}m, 2217 DEGs were identified, of
which 1255 were up-regulated and 962 were down-regulated
(Figure\textbf{8A} ). For \emph{T. laevis} , at \textless{}150\selectlanguage{greek}μ\selectlanguage{english}m, 18,036
DEGs were identified, of which 10,445 were up-regulated and 7591 were
down-regulated. In 150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m anther length,
when\emph{T. laevis} and control libraries were compared, 15,505 DEGs
were generated with 5994 up-regulated and 9511 were down-regulated.
Similarly, at 400\textasciitilde{}700 \selectlanguage{greek}μ\selectlanguage{english}m anther length, 4337 DEGs were
identified, of which 1116 were up-regulated and 3221 were
down-regulated. In 700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m anther length, 7909 DEGs
were identified, of which 3246 were up-regulated and 4663 were
down-regulated, while for 1100\textasciitilde{}1300 \selectlanguage{greek}μ\selectlanguage{english}m length, 15,261
DEGs were identified, of which 7121 were up-regulated and 8140 were
down-regulated (Figure \textbf{8B} ).

\textbf{3.12 Gene ontology (GO) enrichment analysis of DEGs
after\emph{T. controversa} and \emph{T. laevis} infection}

GO categories were developed using the GO database
(\emph{http://www.geneontology.org/}) in order to evaluate potential DEG
functions. GO was categorized into three main domains based on their
functions: biological process, cellular component, and molecular
function. For \emph{T. controversa} , DEGs were classified into
molecular function, cellular component and biological process. Our
results showed that the most significant molecular function enrichments
in the ``binding'' and ``catalytic activity''. In contrast, the most
significant cellular component enrichments occurred in ``membrane
part'', ``cell part'', ``organelle'' and ``membrane'', while biological
process enrichments were in ``metabolic process'' and ``cellular
process'' (Figure \textbf{S4A} ) at various stages of anther
development. For T. \emph{laevis} , results showed that the most
significant molecular function enrichments in the ``catalytic activity''
and ``binding''. In contrast, the most significant cellular component
enrichments occurred in ``cell part'' ``organelle'', ``membrane part''
and ``membrane'', while biological process enrichments were in
``metabolic process'' and ``cellular process'' (Figure \textbf{S4B} ) at
various stages of anther development. When these comparative results of
\emph{T. controversa} and \emph{T. laevis} are illustrated as Venn
diagram, it is clear that both unique and shared DEGs occur between, and
among, pairs (Figure \textbf{S4} ). For \emph{T. controversa} , 1639
DEGs in \textless{}150 \selectlanguage{greek}μ\selectlanguage{english}m anthers, 1706 DEGs in 150\textasciitilde{}400
\selectlanguage{greek}μ\selectlanguage{english}m anthers, 1277 DEGs in 400\textasciitilde{}700 \selectlanguage{greek}μ\selectlanguage{english}m anthers, 709 DEGs in
700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m anthers and 811 DEGs in
1100\textasciitilde{}1300 \selectlanguage{greek}μ\selectlanguage{english}m anthers shared with the others compared
libraries respectively, moreover, 96 DEGs are shared across all
comparisons (Figure \textbf{9A} ). To elucidate the transcriptional
changes occurring after \emph{T. controversa} infection, we demonstrated
the expression pattern by using hierarchical clustering analysis. There
were more up-regulated genes than down-regulated genes in \emph{T.
controversa} -infected plants. At the same time, we found that most of
these genes are related to redox reaction, chitinase, phenylpropane
synthesis and MAPK signal transduction (Figure\textbf{9B} ). For
\emph{T. laevis} , For example, 11420 DEGs in \textless{}150 \selectlanguage{greek}μ\selectlanguage{english}m anthers,
11279 DEGs in 150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m anthers, 3899 DEGs in
400\textasciitilde{}700 \selectlanguage{greek}μ\selectlanguage{english}m anthers, 6315 DEGs in
700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m anthers and 9322 DEGs in
1100\textasciitilde{}1300 \selectlanguage{greek}μ\selectlanguage{english}m anthers shared with the others compared
libraries respectively, moreover, 230 DEGs are shared across all
comparisons (Figure \textbf{9C} ). To elucidate the transcriptional
changes occurring after \emph{T. laevis} infection, we demonstrated the
expression pattern by using hierarchical clustering analysis.
Additionally, the expression patterns of 230 DEGs shared in above five
anthers growth stages were analyzed, and it was found that most genes
were related to stress response, defense response and photosynthesis
(Figure \textbf{9D} ).

\textbf{3.13 KEGG enrichment analysis of DEGs}

KEGG analyses were performed to better understand the molecular
associations among the DEGs. Hundreds of transcripts were differentially
expressed in different anther stages of wheat attacked by \emph{T.
laevis} and \emph{T. controversa} . For \emph{T. laevis} , during
0\textasciitilde{}150 \selectlanguage{greek}µ\selectlanguage{english}m, the top 20 KEGG enrichment pathways were
showed, but topmost pathways were protein processing in endoplasmic
reticulum and plant pathogen interaction (Figure \textbf{S5A} ). For
150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m, the topmost pathways were plant hormone
signal transduction and plant pathogen interaction (Figure\textbf{S5B}
). For 400\textasciitilde{}700 \selectlanguage{greek}μ\selectlanguage{english}m and 700\textasciitilde{}1100 \selectlanguage{greek}µ\selectlanguage{english}m, the
topmost pathway was protein processing in endoplasmic reticulum (Figure
\textbf{S5CD} ). Similarly, for 1100\textasciitilde{}1300 \selectlanguage{greek}µ\selectlanguage{english}m, the
topmost pathways were protein processing in endoplasmic reticulum and
starch and sucrose metabolism (Figure \textbf{S5E} ). For \emph{T.
controversa} , during 0\textasciitilde{}150 \selectlanguage{greek}µ\selectlanguage{english}m, the top 20 KEGG
enrichment pathways were demonstrated, but topmost pathways were
phenylpropanoid biosynthesis, glutathione metabolism, MAPK signaling
pathway plant, protein processing in endoplasmic reticulum and plant
pathogen interaction (Figure\textbf{S5F} ). For 150\textasciitilde{}400
\selectlanguage{greek}μ\selectlanguage{english}m, the topmost pathways were phenylpropanoid biosynthesis, glutathione
metabolism and MAPK signaling pathway plant (Figure \textbf{S5G} ). For
400\textasciitilde{}700 \selectlanguage{greek}μ\selectlanguage{english}m, the topmost pathways were phenylpropanoid
biosynthesis and glutathione metabolism (Figure \textbf{S5H} ). For
700\textasciitilde{}1100 \selectlanguage{greek}µ\selectlanguage{english}m, the topmost pathways were glutathione
metabolism, MAPK signaling pathway plant, amino sugar and nucleotide
sugar metabolism and phenylpropanoid biosynthesis (Figure \textbf{S5I}
). Similarly, for 1100\textasciitilde{}1300 \selectlanguage{greek}µ\selectlanguage{english}m, the topmost pathways
were phenylpropanoid biosynthesis and plant-pathogen interaction (Figure
\textbf{S5J} ).

\textbf{3.14 Expression of pathogenesis-related genes after \emph{T.
laevis} and \emph{T. controversa} infection}

For \emph{T. laevis} , we found that the transcription levels of 81
pathogenesis-related (PR) genes were changed including; 28 PR genes
during 0\textasciitilde{}150 \selectlanguage{greek}μ\selectlanguage{english}m, 8 PR genes during
150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m, 8 PR genes during 400\textasciitilde{}700
\selectlanguage{greek}μ\selectlanguage{english}m, 11 PR genes during 700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m and 25 PR genes
during 1100\textasciitilde{}1300 \selectlanguage{greek}μ\selectlanguage{english}m anthers. For \emph{T. controversa} ,
transcription levels of 52 PR genes were changed including: 13 PR genes
during 0\textasciitilde{}150 \selectlanguage{greek}μ\selectlanguage{english}m, 9 PR genes during
150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m, 16 PR genes during 400\textasciitilde{}700
\selectlanguage{greek}μ\selectlanguage{english}m, 8 PR genes during 700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m and 6 PR genes during
1100\textasciitilde{}1300 \selectlanguage{greek}μ\selectlanguage{english}m anthers (Table \textbf{S3} ).

\textbf{3.15 Expression of anther related genes after \emph{T.
laevis}and \emph{T. controversa} infection}

For \emph{T. laevis} , we found that the transcription levels of 52
anther related genes were changed including; 10 at 0\textasciitilde{}150
\selectlanguage{greek}μ\selectlanguage{english}m, 16 PR at 150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m, 4 at 400\textasciitilde{}700
\selectlanguage{greek}μ\selectlanguage{english}m, 2 at 700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m and 20 at 1100\textasciitilde{}1300
\selectlanguage{greek}μ\selectlanguage{english}m anthers. Similarly, for \emph{T. controversa} , transcription levels
of 54 anther related genes were changed including; 9 at
0\textasciitilde{}150 \selectlanguage{greek}μ\selectlanguage{english}m, 19 at 150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m, 10 at
400\textasciitilde{}700 \selectlanguage{greek}μ\selectlanguage{english}m, 2 at 700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m and 14 at
1100\textasciitilde{}1300 \selectlanguage{greek}μ\selectlanguage{english}m anthers (Table \textbf{S4} ).

\textbf{3.16 Validation of RNA-Seq data}

Total RNA at five developmental stages during both pathogens infection
provided templates for qRT-PCR validation. For \emph{T. laevis} , we
randomly selected 11 DEGs to validate our RNA-Seq results. The qRT-PCR
data for these genes were consistent with the RNA-Seq results from the
five anther lengths, which indicated a high degree of reproducibility
between transcript abundances assayed using RNA-Seq and the expression
profiles revealed by qRT-PCR data (Figure \textbf{S6A} ). For \emph{T.
controversa} , 8 DEGs of different anther lengths were randomly selected
for validation. The RNA-Seq and qRT-PCR analysis results showed high
degree of reproducibility (Figure \textbf{S6B} ).

\textbf{3.17 Knockdown of \emph{TaEC} reduces wheat resistant to\emph{T.
controversa} and \emph{T. laevis}}

The results showed that BSMV:TaEC inoculated leaves has strip like
chlorosis symptoms, while BSMV:\selectlanguage{greek}γ \selectlanguage{english}inoculated leaves have yellowing spots
by virus infection at 13 dpi (Figure 10A-ab).And then, at 30 dpi, more
obvious withering and necrosis symptoms were appeared after BSMV:TaEC
inoculation plants than BSMV:\selectlanguage{greek}γ \selectlanguage{english}inoculation (Figure 10A-cd). The
silencing BSMV:\selectlanguage{greek}γ \selectlanguage{english}(left) and BSMV:TaEC (right) after \emph{T.
controversa} and \emph{T. laevis} inoculation, kernels in spike of
BSMV:TaEC silenced plants showed heavier symptoms than BSMV:\selectlanguage{greek}γ \selectlanguage{english}(Figure
6B-ef).Combined the disease index. data, the results showed that
\emph{TaEC} contribute to the disease resistance of both pathogens.
There is no disease symptoms on kernels after BSMV:\selectlanguage{greek}γ \selectlanguage{english}(left) and
BSMV:TaEC (right) spikes when spikes are not inoculated with \emph{T.
controversa} or \emph{T. laevis} (Figure 10B-g). Lateral and
longitudinal arrangements of kernels showed that kernels are healthy
after only silencing the genes, which not inoculated with \emph{T.
controversa} and \emph{T. laevis} (Figure 10C), respectively. We
performed qRT-PCR to define the \emph{TaEC} transcript levels in the
kernels inoculated with BSMV:\selectlanguage{greek}γ \selectlanguage{english}and BSMV:TaEC at (0, 10, 15, 22, 35 and
42d) intervals after \emph{T. controversa} or \emph{T. laevis}infection.
The results showed that the expression of BSMV:TaEC was significantly
lower than that of BSMV:\selectlanguage{greek}γ \selectlanguage{english}at 0 dpi, indicating that the TaEC gene was
successfully silenced (Figure 10D, E). After inoculation with \emph{T.
controversa} , the expression of TaEC was extremely significantly lower
than that of BSMV:\selectlanguage{greek}γ \selectlanguage{english}at 10 dpi, and increased at 15 dpi, but still
significantly lower than that of BSMV:\selectlanguage{greek}γ \selectlanguage{english}(Figure 10D). There was no
significant difference in the expression of BSMV:TaEC and BSMV:\selectlanguage{greek}γ \selectlanguage{english}TaEC
genes after 22dpi. Similarly, the expression of TaEC transcript was
significantly decreased at 10 dpi and 15 dpi after inoculation with
\emph{T. laevis} , which was showed lower expression level than that of
BSMV:\selectlanguage{greek}γ\selectlanguage{english}(Figure 10E). The grains length in BSMV:TaEC was 83.23 \selectlanguage{ngerman}± 3.62 mm,
while in BSMV:\selectlanguage{greek}γ \selectlanguage{english}was 88.29 \selectlanguage{ngerman}± 3.42 mm. Similarly, grain width in BSMV:TaEC
was 81.18 ± 3.51 mm, while in BSMV:\selectlanguage{greek}γ \selectlanguage{english}was 80.81 \selectlanguage{ngerman}± 2.8 mm.

\textbf{4 DISCUSSION}

The anther development in wheat fallows a pattern similar to other
members of \emph{Poaceae} family, including \emph{Brachypodium
distachyon} (Sharma et al., 2015) and rice (Zhang et al., 2006).
Resemblances also exist in \emph{Arabidopsis thaliana} (Chang et al.,
2011) but some distinct differences were recorded in wheat anther
development. The critical step in the anther development is the balance
of cell sizes (width, length and depth) and the cell counts of EPI, EN,
SPL, ML, TA, PMC, MP and PG cells. Therefore, we investigated the
effects of \emph{T. laevis} and \emph{T. controversa} on above cell
sizes and number on comparison with control anthers.

\textbf{4.1 The timeline of cell development of normal wheat anther}

In this study, we firstly construct the timeline of wheat anther
development using confocal microscopy. The AR cells appear from L2 layer
of anthers \textless{} 150 \selectlanguage{greek}µ\selectlanguage{english}m and differentiate into EN, SPL and PMC
cells of anthers. In this range, vascular tissues developed more
clearly. The SPL cells differentiate into ML and TA almost in 400 \selectlanguage{greek}µ\selectlanguage{english}m
long anthers. PMC cells differentiate into Tds almost in 700 \selectlanguage{greek}µ\selectlanguage{english}m anthers,
where ML start to be wrinkled between TA and EN cells and disappear
almost in 1100 \selectlanguage{greek}µ\selectlanguage{english}m anthers. Tds start to change into MP almost from
720-1000 \selectlanguage{greek}µ\selectlanguage{english}m anthers. Here ML is scarcely present, and TA is at its most
swollen and largest size. However, near to 1200 \selectlanguage{greek}µ\selectlanguage{english}m, MP become vacuolate
towards the TA walls. An exine layer start to develop on the MP and TA
began to reduce in size (Browne et al., 2018). At 1300 \selectlanguage{greek}µ\selectlanguage{english}m, TA cells
disappear, and microspore grow large and began to change in PG cells. At
2300 \selectlanguage{greek}µ\selectlanguage{english}m, mature PG formed and which are starch filled. EPI cells at
stomium start to degrade, causing locules to open and PG start to be
released (Figure \textbf{1} ). \emph{RAFTIN1} and \emph{TaMYB80} play a
crucial role in TA development and TA program cell death (Gao et al.,
2013; Wang et al., 2003; Murray et al., 2003; Millar et al., 2005). We
approved the anther timeline by analyzing the expression of TA
development and TA program cell death in different anther length.
Results showed that the expression of \emph{RAFTAN1} was high in 200-900
\selectlanguage{greek}µ\selectlanguage{english}m anther compared to other sized anthers. While, \emph{TaMYB80} showed
maximum expression in 1100-1300 \selectlanguage{greek}µ\selectlanguage{english}m anthers. The expression results
suggested that TA start to develop from 400 \selectlanguage{greek}µ\selectlanguage{english}m anthers and almost
disappear in 1300 \selectlanguage{greek}µ\selectlanguage{english}m anthers (Figure \textbf{S1} ). Confocal results
showed that PMC cells were surrounded by three somatic cell layers:
namely EPI, EN and SPL from interior to outside (Figure \textbf{2B} ).
Except EPI, the three inner cells were differentiated after mitotic
division in AR cells. Hence, earliest and most important, steps in
anther development are the production of AR cells. The final structure
results in the PMC enclosed by a serious of somatic cells, which are EN,
SPL and EPI (Figure \textbf{2B} ). Finally, after meiosis PMC cells
differentiate into PG almost from 1300 to 2300 \selectlanguage{greek}µ\selectlanguage{english}m, which released from
pollen sac by opening the stomium (Figure \textbf{2I} ). However,
according to Browne et al\emph{.} (2018), AR cells generated primary
parietal cells (PPC) and primary sporogenous cells (PSC) after mitosis.
Then PPC divide into EN and ML and PSC divide into PMC cells, which
produce Tds. In our results, AR cells directly differentiate into EN,
SPL and PMC cells and then SPL cells divide into ML and TA and PMC cells
divide into Tds that differentiate into MP and PG in short time.
Moreover, the difference in wheat anther development was noted from
model plant, \emph{Arabidopsis thaliana} (Chang et al., 2011). However,
according to Zhang and Wilson (2009) the EN, SPL and PMC cells
differentiate from AR cells then it differentiate into other cells.

\textbf{4.2 Infection of \emph{T. laevis} and \emph{T. controversa} in
anther cells}

The common, dwarf, and other related bunt fungi have a long infection
and development process from seedling to grain filling (Lu et al., 2015;
Nguyen et al., 2019; Bokore et al., 2019). In fact, the mycelium that
develops in the infected seedling remains spares until a massive
proliferation in floral tissue (Ghareeb et al., 2011). In the present
study, we inoculated wheat spikes with mycelia of \emph{T. laevis}
and\emph{T. controversa} to study the effects of infection on the
development of wheat anthers. We tracked the prevalence of \emph{T.
laevis} or \emph{T. controversa} mycelia into somatic and reproductive
anther cells for understanding the process of infection. Hyphae
proliferate finally into the pollen grains and releasing of these
infected pollens for pollination, is critical for normal plant
reproduction and produced seeds is transformed into a generally
spherical sorus termed a bunt ball that contains dark-colored mass of
teliospores (Goates \& Peterson, 1993). Our results showed that hyphae
of \emph{T. laevis} was well established on EPI cells at 800 \selectlanguage{greek}µ\selectlanguage{english}m long and
rupture the cell structure (Figure \textbf{4A-C} ). EPI cells are the
most earliest infected anther cells from filaments, from which the
hyphae spread to other connective cells. Thus, the nearest next cell of
EPI is EN, which was infected at 1220 \selectlanguage{greek}µ\selectlanguage{english}m long anther. Similarly, TA and
PG cells were infected at 1300 \selectlanguage{greek}µ\selectlanguage{english}m and 1520 \selectlanguage{greek}µ\selectlanguage{english}m. By comparison with
control anthers, TA should be disappeared at 1300 \selectlanguage{greek}µ\selectlanguage{english}m, but which was
visible at 1300 \selectlanguage{greek}µ\selectlanguage{english}m (Figure \textbf{4G-I).} We conclude that the pathogen
prevents normal development of TA and PG which interrupts the production
of signals that results in the development of reproductive cells (Walbot
\& Skibbe, 2010; Li, 2013). The mechanism of \emph{T.
controversa}infection was similar to that of \emph{T. laevis} , but
delayed differentiation in TA and PG compared to control. TA was
observed at 2100 \selectlanguage{greek}µ\selectlanguage{english}m and PG was also present on the 2100 \selectlanguage{greek}µ\selectlanguage{english}m long anthers
(Figure\textbf{6G-I} ) (Hu et al., 2002; Schirawski et al., 2007).

\textbf{4.3 Effects of \emph{T. laevis} and \emph{T. controversa} on
wheat anther development}

To understand the infection between somatic and reproductive cell in
wheat anther, cell number and size were estimated in infected anthers.
In the case of the \emph{T. laevis} and \emph{T. controversa} wheat
interaction, the consequence of successful infection is the change of
the cell count and cell size of anther. Thus, the normal development of
anther cell layers influence by pathogens (Walbot \& Skibbe, 2010; Li,
2013; Banuett et al., 1996). Confocal imaging allowed measurement of
wheat anther cell counts and sizes after fungal infection during anther
development. By comparing with control anthers, cell size and numbers
increased or decreased significantly due to the infection of \emph{T.
laevis} or \emph{T. controversa} (Figures \textbf{5, 7,}
Tables\textbf{3, 4, 5, 6} )\emph{.} Anther consists of unreproductive
and reproductive cell types, and almost each cell type showed
alterations in width, length and depth, and /or volume and percentage in
some stages after infection. Our results showed that cell count of EPI,
EN and ML significantly decreased, but no statistically observation was
noted in the cell sizes of EPI, EN, and ML after \emph{T. laevis}
infection along Y-axis. In the case of \emph{T. controversa} the cell
count of EPI along X- and Y- axes increased at the first stage of
development, which suggest abnormalities in the normal process of
development. The SPL cells produce ML and TA, after infection the cell
count of X*Y in ML and TA increased, mean \emph{T. controversa} disrupts
the normal development of both cells (Figure \textbf{7C,} Table
\textbf{5} ). Additionally, the all-somatic and reproductive cells of
anthers affected by the \emph{T. controversa} . However, the volume of
ML and TA cells decreased in 400-700 \selectlanguage{greek}µ\selectlanguage{english}m sized anthers, which infect the
formation process of MP and PG. The cell width, length and volume of PMC
and MP cells increased in 400-700 \selectlanguage{greek}µ\selectlanguage{english}m and 700-1100 \selectlanguage{greek}µ\selectlanguage{english}m anthers compared to
control plants (Kelliher \& Walbot, 2011). The infected TA cells remain
undifferentiated for long time compared to normal cells. The cell
counts, and cell sizes of cell types changed by both fungal infection.

\textbf{4.4 RNA-Seq data in response to \emph{T. laevis} and \emph{T.
controversa}}

Pathogenesis-related (PR) genes and WRKY transcription factors play
important role in defense mechanism and in regulation of defense genes
(Anguelova-Merhar et al., 2001; Sels et al., 2008; Pandey \& Somssich,
2009; Rushton et al., 2010; Bai et al., 2018). We compared the
transcription level between both pathogens infected and control plants
at different wheat anther stages. Our results showed that
transcriptional levels of 81 PR genes after \emph{T. laevis} and 52 PR
genes after \emph{T. controversa} were changed. UDT1 is a transcription
factor that plays a key role in the development of the tapetum and is
required for anther male fertility and pollen differentiation (v et al.,
2005; Liu et al., 2010). Additionally, the transcription factor TDR is
involved in the regulation of programmed cell death (PCD) of tapetum and
degradation of male reproductive development (Li et al., 2006). TDR
plays an upstream role in the regulation of tapetum PCD and interacts
with EAT1/DTD (Niu et al., 2013) and may also play a regulatory role in
lipid metabolism involved in pollen wall formation (Zhang et al., 2008).
In this study, the transcription factors UDT1, TDR and EAT1 were
expressed at all stages of anthers development and TDR mostly
down-regulated at fifth stage (1100-1300 \selectlanguage{greek}µ\selectlanguage{english}m) support hypothesis that
tapetum cells degraded in the last stages. Polygalacturonase is a cell
wall hydrolytic enzyme, which is related to various cell separation
processes during plant development including; seed germination, anther
dehiscence, organ shedding and fruit ripening (Kim \& Patterson, 2006;
Ogawa et al., 2009). In our study, the expression of polygalacturonase
gene was down regulated at all stages of anther development (700-1100
\selectlanguage{greek}µ\selectlanguage{english}m), indicating that both pathogens alters the normal structure of
anther development as a result anthers lose their pollination
activities.

According to GO enrichment analysis of differentially expressed genes in
control and both pathogens infected libraries, the results showed that
binding, catalytic activity, membrane part, cell part, metabolic process
and cellular process were had the highest number of DEGs during plant
pathogen interaction (Figure \textbf{S4} ). GO enrichment analysis
suggest that both pathogens alters the morpho-physiological and defense
mechanisms of the plants (Berger et al., 2007). KEGG is database
resource for understanding high level functions and utilities of a
biological system. This is especially useful for large scale molecular
datasets generated by genome sequencing and other high-throughput
experiment technologies, providing valuable classification for studies
of biological complex process (Kanehisa \& Goto, 2000). KEGG enrichment
analysis showed that metabolic and cellular pathways were expressed
significantly at all above anther stages after \emph{T. laevis}
and\emph{T. controversa} infection. The photosynthesis is a basic
physiological processes of plant growth (Deng et al., 2009). The results
obtained from KEGG analysis of DEGs showed that genes involved
photosynthesis significantly changed after \emph{T. laevis} and \emph{T.
controversa} infection (Figure \textbf{S5} ). Phenylalanine metabolism
are the substrate molecules of primary and secondary metabolic pathways
and have role in the viability of all cells (Adams et al., 2019).
Endoplasmic reticulum are important organelle and have a key role for
protein production destined for the plasma membrane, vacuoles and
apoplast in plants (Eichmann \& Sch\selectlanguage{ngerman}äfer, 2012). Plants hormones are the
key regulators for many functions including primary (plant growth and
development) and secondary (induction of defence programme) (Bolwell,
1997; Yvon \& Joanne, 2011). Starch and sucrose have a central role in
the carbon budget of the majority of plants, and plays a dual role in
carbon allocation (MacNeill et al., 2017). Glycolysis have a major role
in respiration rate of the plants by producing the substrates that are
essential for respiration (Megguer et al., 2017). Our results showed
that KEGG analysis of DEGs showed that the genes involved in
phenylalanine metabolism, endoplasmic reticulum, plant hormone signal
transduction, starch, sucrose and glycolysis were significantly changed
after \emph{T. laevis} and \emph{T. controversa} infection in different
stages of anther development (Figure \textbf{S5} ).

\textbf{4.5 \emph{TaEC} contribute to wheat resistance to bunt pathogens
based on VIGS}

To characterize in more detail the role of \emph{TaEC} during wheat
response to bunt pathogens, BSMV-VIGS, which has been applied to
analysis gene function in wheat and barley, was used (Gao et al., 2013).
This is the first time to silence \emph{TaEC} through BSMV-based VIGS in
wheat spikes against bunt pathogens. VIGS was done against \emph{Apple
latent spherical virus} (CMV) and \emph{Cucumber mosaic virus} (ALSV) in
soybean at different stages of growth development (Nagamatsu et al.,
2007; Yamagishi \& Yoshikawa, 2009), inoculation of recombinant virus on
different parts of the plant. We first targeted the \emph{TaEC} genes in
wheat. These results confirmed that VIGS successfully established in
wheat crops against \emph{T. controversa} and \emph{T. laevis}infection.
Additionally, provide a genome wide gene expression profile for wheat
crops infected by \emph{T. laevis} and \emph{T. controversa}and may help
to explore the regulatory mechanisms governing the response of wheat
crops to these pathogens. According to our knowledge, it is the first
study to build up the timeline of wheat anther and reveal the effects of
both pathogens on wheat anther development and transcriptome alterations
by confocal analysis and RNA-seq, and additionally, firstly
establishment of VIGS to explore the gene function after infection of
two bunt pathogens.

\textbf{Acknowledgments}

We thank the Institute of Genetics and Developmental Biology, Chinses
Academy of Sciences, for providing the confocal microscopy. This work
was supported by Natural Science Foundation of China (31761143011 and
31571965).

\textbf{Author contribution}

LG designed the experiments, write the manuscript and supervised the
project. MF, DC and TH performed the experiment. YY, QG and WC provided
some suggestions for the editing the manuscript.

\textbf{Conflict of interest}

Authors declare no conflict of interest between them

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\textbf{Figure legends}

\textbf{Figure 1.} Formation of wheat anther cell layers. The anther
primordium only contains L1, L2 and L3 layers. Some cells from L2 become
archesporial (AR) cells \textless{} 150 \selectlanguage{greek}µ\selectlanguage{english}m, which differentiate into
epidermis (EN), secondary parietal layer (SPL) and pollen mother cells
(PMC) \textgreater{} 150 \selectlanguage{greek}µ\selectlanguage{english}m anthers. Then SPL divides to form middle
layer (ML) and tapetum (TA) almost at 400 \selectlanguage{greek}µ\selectlanguage{english}m. Subsequently, PMC form the
MP at 700 \selectlanguage{greek}µ\selectlanguage{english}m, which differentiate to pollen grain (PG) almost at 1300
\selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{Figure 2.} Confocal images of anther developmental
cells.\textbf{(A)} The EPI cells have obvious shape and AR cells began
to form at 100 \selectlanguage{greek}µ\selectlanguage{english}m. \textbf{(B)} At 310 \selectlanguage{greek}µ\selectlanguage{english}m AR cells differentiate into
EN, SPL and PMC cells after mitosis. \textbf{(C)} EN cells clearly
defined, which have oval shape at 580 \selectlanguage{greek}µ\selectlanguage{english}m. \textbf{(D)} SPL are round at
380 \selectlanguage{greek}µ\selectlanguage{english}m.\textbf{(E)} PMC cells have round shape and almost have clear
structure at 370 \selectlanguage{greek}µ\selectlanguage{english}m. \textbf{(F, G)} Tds formed after meiosis in PMC
cells at 660 and 680 \selectlanguage{greek}µ\selectlanguage{english}m, respectively. PMC cells totally disappear, and
ML cell began to crush \textbf{(H)} Tds differentiate into MP at 700 \selectlanguage{greek}µ\selectlanguage{english}m
and ML become disappear at almost 1100 \selectlanguage{greek}µ\selectlanguage{english}m and MP grow large create space
from TA cells\textbf{(I)} PG become round at 1630 \selectlanguage{greek}µ\selectlanguage{english}m and gametes develop
within pollen grains and ML, TA, MP disappear after PG development.
Scale bar: a, c, d, f, g = 25 \selectlanguage{greek}µ\selectlanguage{english}m, b, e, I = 50 \selectlanguage{greek}µ\selectlanguage{english}m, h= 75 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{Figure 3.} The cell numbers and cell sizes of control wheat
anther was measured using velocity software. \textbf{(A)} Cell count of
anther layers along X-axis. \textbf{(B)} Cell count of anther layers
along Y-axis. \textbf{(C)} Total cells/locule (X*Y-tips) of anther
layers. \textbf{(D)} Percentage of locule cell count of anther
layers.\textbf{(E)} Cell width of anther layers along X-axis.
\textbf{(F)} Cell length of anther layers along Y-axis. \textbf{(G)}
Cell depth of anther layers along Z-axis. \textbf{(H)} Cell volume of
anther layers was derived by multiplying the width, length and depth
\textbf{(I)} Y/X value of anther layers were measured by dividing the
length and width of anther layers \textbf{(J)} Percentage of locule
volume of anther layers. The cell counts, and sizes were quantified with
at least 60 anthers per stage.

\textbf{Figure 4.} \emph{T. laevis} location on anther developmental
cells of wheat anther. \textbf{(A-C)} Hyphae on EPI cells (800
\selectlanguage{greek}µ\selectlanguage{english}m);\textbf{(D-F)} Hyphae on EN cells (1220 \selectlanguage{greek}µ\selectlanguage{english}m); \textbf{(G-I)} Hyphae
on TA cells (1300 \selectlanguage{greek}µ\selectlanguage{english}m) \textbf{(J-L)} Hyphae on mature PG (1520 \selectlanguage{greek}µ\selectlanguage{english}m).
Scale bars: a-c = 50 \selectlanguage{greek}µ\selectlanguage{english}m; d-f = 25 \selectlanguage{greek}µ\selectlanguage{english}m; g-i = 10 \selectlanguage{greek}µ\selectlanguage{english}m; j-l = 25 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{Figure 5.} The cell numbers and cell sizes of \emph{T.
laevis}infected wheat anther was measured using velocity software.
\textbf{(A)}Cell count of anther layers along X-axis\textbf{. (B)} Cell
count of anther layers along Y-axis. \textbf{(C)} Total cells/locule
(X*Y-tips) of anther layers. \textbf{(D)} Percentage of locule cell
count of anther layers. \textbf{(E)} Cell width of anther layers along
X-axis.\textbf{(F)} Cell length of anther layers along Y-axis.
\textbf{(G)}Cell depth of anther layers along Z-axis. \textbf{(H)} Cell
volume of anther layers was derived by multiplying the width, length and
depth\textbf{(I)} Y/X value of anther layers were measured by dividing
the length and width of anther layers \textbf{(J)} Percentage of locule
volume of anther layers. The cell counts, and sizes were quantified with
at least 60 anthers per stage.

\textbf{Figure 6.} \emph{T. controversa} location on anther
developmental cells of wheat anther. \textbf{(A-C)} Hyphae on EPI cells
1000 (\selectlanguage{greek}µ\selectlanguage{english}m); \textbf{(D-F)} Hyphae on EN cells (1300 \selectlanguage{greek}µ\selectlanguage{english}m);
\textbf{(G-I)}Hyphae on TA cells (2100 \selectlanguage{greek}µ\selectlanguage{english}m) \textbf{(J-L)} Hyphae on
mature PG (2100 \selectlanguage{greek}µ\selectlanguage{english}m). Scale bars: a-c = 50 \selectlanguage{greek}µ\selectlanguage{english}m; d-l= 25 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{Figure 7.} The cell numbers and cell sizes of \emph{T.
controversa} infected wheat anther was measured using velocity
software.\textbf{(A)} Cell count of anther layers along X-axis.
\textbf{(B)} Cell count of anther layers along Y-axis. \textbf{(C)}
Total cells/locule (X*Y-tips) of anther layers. \textbf{(D)} Percentage
of locule cell count of anther layers. \textbf{(E)} Cell width of anther
layers along X-axis. \textbf{(F)} Cell length of anther layers along
Y-axis.\textbf{(G)} Cell depth of anther layers along Z-axis.
\textbf{(H)} Cell volume of anther layers was derived by multiplying the
width, length and depth \textbf{(I)} Y/X value of anther layers were
measured by dividing the length and width of anther layers \textbf{(J)}
Percentage of locule volume of anther layers. The cell counts, and sizes
were quantified with at least 60 anthers per stage.

\textbf{Figure 8} . The DEGs between \emph{T. controversa} and \emph{T.
laevis} samples. (\textbf{A} ) Numbers of DEGs compared between \emph{T.
laevis} and control samples. (\textbf{B} ) Numbers of DEGs compared
between \emph{T. controversa} and control samples. DEGs are shown in
dark (up-regulated) and light white (down-regulated). Control\_1\_vs\_
infected 1\_G = 0\textasciitilde{}150 \selectlanguage{greek}μ\selectlanguage{english}m, control\_2\_vs\_ infected
2\_G= 150\textasciitilde{}400 \selectlanguage{greek}μ\selectlanguage{english}m, control\_3\_vs\_ infected 3\_G =
400\textasciitilde{}700 \selectlanguage{greek}μ\selectlanguage{english}m, control\_4\_vs\_ infected 4\_G
=700\textasciitilde{}1100 \selectlanguage{greek}μ\selectlanguage{english}m and control\_5\_vs\_ infected
5\_G=1100\textasciitilde{}1300 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{Figure 9.} Analysis of differentially expressed genes in
different anther length stages. (\textbf{a} ) Venn diagram analysis of
the DEGs in different anther length stages after inoculation
with\emph{T. laevis} . (\textbf{b} ) Hierarchical clustering heatmap of
expression profiles of selected DEGs in response to \emph{T.
laevis}infection. (\textbf{c} ) Venn diagram analysis of the DEGs in
different anther length stages after inoculation with \emph{T.
controversa} . (\textbf{d} ) Hierarchical clustering heatmap of
expression profiles of selected DEGs in response to \emph{T.
controversa} infection. Each column shows a library, and each row shows
DEG expression. The colors blue, white and red indicate low, medium, and
high expression patterns of genes, respectively.

\textbf{Figure 10.} \emph{TaEC} increase wheat resistance to bunt
pathogens. (A) \textbf{a} : The wheat leaves inoculated with BSMV: TaEC
at 13 days post inoculation (dpi). \textbf{b:} The wheat leaves
inoculated with BSMV: \selectlanguage{greek}γ \selectlanguage{english}at 13 dpi. \textbf{c} : The wheat leaves
inoculated with BSMV:TaEC at 30 (dpi). \textbf{d} : The wheat leaves
inoculated with BSMV:\selectlanguage{greek}γ \selectlanguage{english}at 30 (dpi). (\textbf{B} ) e: The kernels
inoculated with BSMV:\selectlanguage{greek}γ \selectlanguage{english}(left) and BSMV:TaEC (right) after \emph{T.
controversa} inoculation \textbf{f} : The kernels inoculated with BSMV:\selectlanguage{greek}γ
\selectlanguage{english}(left) and BSMV:TaEC (right) after \emph{T. laevis}
inoculation.\textbf{g} : The kernels only infected with BSMV:\selectlanguage{greek}γ\selectlanguage{english}(left) and
BSMV:TaEC (right) without inoculation of \emph{T. controversa} and
\emph{T. laevis} . (C) \textbf{h} : Lateral arrangement of wheat grains
infected with BSMV:\selectlanguage{greek}γ \selectlanguage{english}and BSMV:TaEC. \textbf{i} : Longitudinal
arrangement of wheat grains infected with BSMV:\selectlanguage{greek}γ \selectlanguage{english}and BSMV:TaEC.
(\textbf{D} ) The relative expression level of \emph{TaEC} after
\emph{T. controversa} inoculation. (\textbf{E} ) The relative expression
level of \emph{TaEC} after \emph{T. laevis} inoculation.

\textbf{Table}

\textbf{Table 1.} Cell counts of anther in control samples. X-indicates
the cell counts along X-axis, Y-axis indicate cell counts along Y-axis,
X*Y is the total cell count per locule and percent mean percentage of
all cells of epidermis (EPI), endothecium (EN), secondary parietal cell
(SPL), middle layer (ML), tapetum (TA), pollen mother cell (PMC),
microspore (MP) and pollen grain (PG). - Indicate that cell is not
appear.

\textbf{Table 2.} Cell sizes and volume of anther in control samples.
X-axis indicates the cell width, Y-axis cell length, and Z-axis cell
depth of epidermis (EPI), endothecium (EN), secondary parietal cell
(SPL), middle layer (ML), tapetum (TA), pollen mother cell (PMC),
microspore (MP) and pollen grain (PG). Cell volume was obtained by
multiplication of X, Y and Z-axes. - Indicate that cell is not appear.

\textbf{Table 3.} Cell counts in \emph{T. laevis} infected anthers.
X-indicates the cell counts along X-axis, Y-axis indicate cell number
along Y-axis, X*Y is the total cell count per locule and percent mean
percentage of all cells of epidermis (EPI), endothecium (EN), secondary
parietal cell (SPL), middle layer (ML), tapetum (TA), pollen mother cell
(PMC), microspore (MP) and pollen grain (PG). Asterisks* indicate
statistically significant differences. (Student's \emph{t-test}
:\emph{*P}  [?] 0.05, \emph{**P}  [?] 0.01). The values without Asterisks*
indicated the statistically non-significant. \selectlanguage{ngerman}± showed standard error. --
indicated that cells not appeared.

\textbf{Table 4.} Cell sizes and volume of anther cells in \emph{T.
laevis} infected anthers. X-axis indicates the cell width, Y-axis cell
length, and Z-axis cell depth of epidermis (EPI), endothecium (EN),
secondary parietal cell (SPL), middle layer (ML), tapetum (TA), pollen
mother cell (PMC), microspore (MP) and pollen grain (PG). Cell volume
was obtained by multiplication of X, Y and Z-axes. Asterisks* indicate
statistically significant differences. (Student's \emph{t-test}
:\emph{*P} \selectlanguage{english} \selectlanguage{english}[?]\selectlanguage{english} 0.05, **\emph{P} \selectlanguage{english} \selectlanguage{english}[?]\selectlanguage{english} 0.01). The values without Asterisks*
indicated the statistically non-significant. +- showed standard error. --
indicated that cells not appeared.

\textbf{Table 5.} Cell counts in \emph{T. controversa} infected anthers.
X-indicates the cell counts along X-axis, Y-axis indicate cell number
along Y-axis, X*Y is the total cell count per locule and percent mean
percentage of all cells of epidermis (EPI), endothecium (EN), secondary
parietal cell (SPL), middle layer (ML), tapetum (TA), pollen mother cell
(PMC), microspore (MP) and pollen grain (PG). Asterisks* indicate
statistically significant differences. (Student's \emph{t-test}
:\emph{*P} \selectlanguage{english} \selectlanguage{english}[?]\selectlanguage{english} 0.05, \emph{**P} \selectlanguage{english} \selectlanguage{english}[?]\selectlanguage{english} 0.01). The values without Asterisks*
indicated the statistically non-significant. +-showed standard error. --
indicated that cells not appeared.

\textbf{Table 6.} Cell sizes and volume of anther cells in \emph{T.
controversa} infected anthers. X-axis indicates the cell width, Y-axis
cell length, and Z-axis cell depth of epidermis (EPI), endothecium (EN),
secondary parietal cell (SPL), middle layer (ML), tapetum (TA), pollen
mother cell (PMC), microspore (MP) and pollen grain (PG). Cell volume
obtained by multiplication of X, Y and Z-axes. Asterisks* indicate
statistically significant differences. (Student's \emph{t-test} :
*\emph{P} \selectlanguage{english} \selectlanguage{english}[?] 0.05, **\emph{P} \selectlanguage{english} \selectlanguage{english}[?]\selectlanguage{english} 0.01). The values without Asterisks*
indicated the statistically non-significant. +- showed standard error. --
indicated that cells not appeared.

Figure 1\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure
2\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 3\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 4\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Figure
5\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Figure 6\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Figure 7\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Figure 8\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

Figure 9\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

Figure 10\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/imagf0/imagf0}

\end{center}
\end{figure}

\textbf{Table 1.} Cell counts of anther cells in control samples.
X-indicates the cell counts along X-axis, Y-axis indicate cell counts
along Y-axis, X*Y is the total cell count per locule and percent mean
percentage of all cells of epidermis (EPI), endothecium (EN), secondary
parietal cell (SPL), middle layer (ML), tapetum (TA), pollen mother cell
(PMC), microspore (MP) and pollen grain (PG). - Indicate that cells are
not appear. \selectlanguage{ngerman}± showed the standard errors.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & 0-150 & 150-400 & 400-700 &
700-1100 & 1100-1300 & 1300-2300\tabularnewline
EPI & X & 7.00\selectlanguage{ngerman}±0.30 & 18.00\selectlanguage{ngerman}±1.41 & 19.50\selectlanguage{ngerman}±1.66 & 22.00\selectlanguage{ngerman}±1.10 & 23.33\selectlanguage{ngerman}±0.18
& 24.83\selectlanguage{ngerman}±0.71\tabularnewline
& Y & 4.00\selectlanguage{ngerman}±0.40 & 16.00\selectlanguage{ngerman}±1.08 & 23.16\selectlanguage{ngerman}±2.41 & 31.56\selectlanguage{ngerman}±2.20 & 34.33\selectlanguage{ngerman}±0.32 &
39.18\selectlanguage{ngerman}±5.74\tabularnewline
& X*Y & 28.00\selectlanguage{ngerman}±0.30 & 288.00\selectlanguage{ngerman}±2.86 & 458.87\selectlanguage{ngerman}±5.64 & 693.75\selectlanguage{ngerman}±5.79 &
801.67\selectlanguage{ngerman}±1.00 & 972.82\selectlanguage{ngerman}±4.57\tabularnewline
& percent & 0.34\selectlanguage{ngerman}±0.15 & 0.53\selectlanguage{ngerman}±0.32 & 0.43\selectlanguage{ngerman}±0.10 & 0.33\selectlanguage{ngerman}±0.81 & 0.32\selectlanguage{ngerman}±0.36 &
0.37\selectlanguage{ngerman}±0.33\tabularnewline
EN & X & 6.05\selectlanguage{ngerman}±1.08 & 7.50\selectlanguage{ngerman}±0.98 & 10.03\selectlanguage{ngerman}±0.82 & 11.50\selectlanguage{ngerman}±0.71 & 12.67\selectlanguage{ngerman}±0.25 &
13.84\selectlanguage{ngerman}±0.76\tabularnewline
& Y & 9.16\selectlanguage{ngerman}±0.35 & 16.00\selectlanguage{ngerman}±2.50 & 43.04\selectlanguage{ngerman}±10.07 & 93.33\selectlanguage{ngerman}±7.94 & 111.33\selectlanguage{ngerman}±0.35 &
115.63\selectlanguage{ngerman}±2.32\tabularnewline
& X*Y & 54.00\selectlanguage{ngerman}±0.78 & 130.50\selectlanguage{ngerman}±2.80 & 411.75\selectlanguage{ngerman}±6.45 & 1076.33\selectlanguage{ngerman}±1.17 &
1411.50\selectlanguage{ngerman}±1.00 & 1595.64\selectlanguage{ngerman}±5.06\tabularnewline
& percent & 0.66\selectlanguage{ngerman}±0.13 & 0.24\selectlanguage{ngerman}±0.27 & 0.36\selectlanguage{ngerman}±0.30 & 0.48\selectlanguage{ngerman}±0.32 & 0.56\selectlanguage{ngerman}±0.53 &
0.61\selectlanguage{ngerman}±0.34\tabularnewline
SPL & X & - & 4.85\selectlanguage{ngerman}±0.87 & - & - & - & -\tabularnewline
& Y & - & 12.10\selectlanguage{ngerman}±1.59 & - & - & - & -\tabularnewline
& X*Y & - & 58.36\selectlanguage{ngerman}±2.02 & - & - & - & -\tabularnewline
& percent & - & 0.14\selectlanguage{ngerman}±0.28 & - & - & - & -\tabularnewline
ML & X & - & - & 5.00\selectlanguage{ngerman}±0.30 & 6.75\selectlanguage{ngerman}±0.84 & - & -\tabularnewline
& Y & - & - & 12.50\selectlanguage{ngerman}±0.32 & 25.03\selectlanguage{ngerman}±6.75 & - & -\tabularnewline
& X*Y & - & - & 57.00\selectlanguage{ngerman}±0.30 & 173.75\selectlanguage{ngerman}±3.67 & - & -\tabularnewline
& percent & - & - & 0.06\selectlanguage{ngerman}±0.37 & 0.08\selectlanguage{ngerman}±0.51 & - & -\tabularnewline
TA & X & - & - & 5.00\selectlanguage{ngerman}±0.35 & 7.33\selectlanguage{ngerman}±1.22 & 8.70\selectlanguage{ngerman}±1.15 & -\tabularnewline
& Y & - & - & 11.50\selectlanguage{ngerman}±0.31 & 24.00\selectlanguage{ngerman}±1.25 & 28.50\selectlanguage{ngerman}±4.51 & -\tabularnewline
& X*Y & - & - & 61.00\selectlanguage{ngerman}±0.50 & 179.78\selectlanguage{ngerman}±1.10 & 245.75\selectlanguage{ngerman}±3.64 &
-\tabularnewline
& percent & - & - & 0.06\selectlanguage{ngerman}±0.50 & 0.08\selectlanguage{ngerman}±0.46 & 0.10\selectlanguage{ngerman}±0.25 & -\tabularnewline
PMC & X & - & 2.00\selectlanguage{ngerman}±0.45 & 3.05\selectlanguage{ngerman}±0.22 & - & - & -\tabularnewline
& Y & - & 4.50\selectlanguage{ngerman}±1.79 & 9.12\selectlanguage{ngerman}±1.92 & - & - & -\tabularnewline
& X*Y & - & 9.00\selectlanguage{ngerman}±7.12 & 30.08\selectlanguage{ngerman}±5.18 & - & - & -\tabularnewline
& percent & - & 0.03\selectlanguage{ngerman}±0.30 & 0.02\selectlanguage{ngerman}±0.61 & - & - & -\tabularnewline
MP & X & - & - & - & 4.03\selectlanguage{ngerman}±1.00 & 4.13\selectlanguage{ngerman}±0.16 & -\tabularnewline
& Y & - & - & - & 12.25\selectlanguage{ngerman}±1.15 & 13.00\selectlanguage{ngerman}±0.40 & -\tabularnewline
& X*Y & - & - & - & 49.32\selectlanguage{ngerman}±2.31 & 53.73\selectlanguage{ngerman}±0.45 & -\tabularnewline
& percent & - & - & - & 0.02\selectlanguage{ngerman}±0.24 & 0.02\selectlanguage{ngerman}±0.39 & -\tabularnewline
PG & X & - & - & - & - & - & 4.38\selectlanguage{ngerman}±1.06\tabularnewline
& Y & - & - & - & - & - & 13.48\selectlanguage{ngerman}±1.14\tabularnewline
& X*Y & - & - & - & - & - & 59.08\selectlanguage{ngerman}±2.28\tabularnewline
& percent & - & - & - & - & - & 0.02\selectlanguage{ngerman}±0.17\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Table 2.} Cell sizes and volume of anther cells in control
samples. X-axis indicates the cell width, Y-axis cell length, and Z-axis
cell depth of epidermis (EPI), endothecium (EN), secondary parietal cell
(SPL), middle layer (ML), tapetum (TA), pollen mother cell (PMC),
microspore (MP) and pollen grain (PG). Cell volume was obtained by
multiplication of X, Y and Z-axes. \selectlanguage{english}- Indicate that cells are not
appear. +- showed the standard error.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & 0-150 & 150-400 & 400-700 &
700-1100 & 1100-1300 & 1300-2300\tabularnewline
EPI & X & 8.00\selectlanguage{ngerman}±1.37 & 8.46\selectlanguage{ngerman}±2.33 & 9.19\selectlanguage{ngerman}±1.64 & 10.29\selectlanguage{ngerman}±2.97 & 12.52\selectlanguage{ngerman}±2.45 &
14.01\selectlanguage{ngerman}± 4.29\tabularnewline
& Y & 16.00\selectlanguage{ngerman}±4.13 & 16.75\selectlanguage{ngerman}±12.45 & 21.25\selectlanguage{ngerman}±3.22 & 34.63\selectlanguage{ngerman}±11.32 & 47.10\selectlanguage{ngerman}±8.15 &
51.08\selectlanguage{ngerman}±9.80\tabularnewline
& Z & 6.67\selectlanguage{ngerman}±3.01 & 6.78\selectlanguage{ngerman}±1.69 & 7.07\selectlanguage{ngerman}±0.33 & 7.79\selectlanguage{ngerman}±1.39 & 9.71\selectlanguage{ngerman}±3.54 &
11.98\selectlanguage{ngerman}±2.13\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & 853.76\selectlanguage{ngerman}±2.37 & 1074.32\selectlanguage{ngerman}±1.13 &
1395.68\selectlanguage{ngerman}±1.36 & 2775.91\selectlanguage{ngerman}±1.65 & 5725.91\selectlanguage{ngerman}±1.38 & 8573.27\selectlanguage{ngerman}±2.26\tabularnewline
& Y/X & 2.00\selectlanguage{ngerman}±0.36 & 1.98\selectlanguage{ngerman}±0.12 & 2.30\selectlanguage{ngerman}±0.39 & 3.33\selectlanguage{ngerman}±0.41 & 3.77\selectlanguage{ngerman}±0.08 &
3.64\selectlanguage{ngerman}±0.95\tabularnewline
& percentage & 0.51\selectlanguage{ngerman}±0.11 & 0.24\selectlanguage{ngerman}±0.30 & 0.25\selectlanguage{ngerman}±0.52 & 0.33\selectlanguage{ngerman}±0.37 & 0.39\selectlanguage{ngerman}±0.10
& 0.27\selectlanguage{ngerman}±0.37\tabularnewline
EN & X & 10.21\selectlanguage{ngerman}±1.78 & 12.67\selectlanguage{ngerman}±3.69 & 18.65\selectlanguage{ngerman}±5.36 & 23.35\selectlanguage{ngerman}±9.75 & 27.91\selectlanguage{ngerman}±8.42
& 30.82\selectlanguage{ngerman}±11.51\tabularnewline
& Y & 8.03\selectlanguage{ngerman}±1.89 & 8.75\selectlanguage{ngerman}±1.69 & 9.75\selectlanguage{ngerman}±1.52 & 10.40\selectlanguage{ngerman}±2.15 & 10.77\selectlanguage{ngerman}±1.39 &
13.54\selectlanguage{ngerman}±4.81\tabularnewline
& Z & 5.22\selectlanguage{ngerman}±0.26 & 5.47\selectlanguage{ngerman}±1.21 & 6.58\selectlanguage{ngerman}±0.27 & 7.55\selectlanguage{ngerman}±0.51 & 8.09\selectlanguage{ngerman}±1.05 &
9.23\selectlanguage{ngerman}±1.67\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & 427.97\selectlanguage{ngerman}±1.35 & 606.42\selectlanguage{ngerman}±0.57 &
1196.49\selectlanguage{ngerman}±1.75 & 1833.44\selectlanguage{ngerman}±1.46 & 2431.78\selectlanguage{ngerman}±2.33 & 3851.70\selectlanguage{ngerman}±3.38\tabularnewline
& Y/X & 0.79\selectlanguage{ngerman}±0.92 & 0.69\selectlanguage{ngerman}±0.35 & 0.52\selectlanguage{ngerman}±0.13 & 0.45\selectlanguage{ngerman}±0.47 & 0.39\selectlanguage{ngerman}±0.79 &
0.44\selectlanguage{ngerman}±0.34\tabularnewline
& percentage & 0.49\selectlanguage{ngerman}±0.98 & 0.14\selectlanguage{ngerman}±0.82 & 0.22\selectlanguage{ngerman}±0.35 & 0.23\selectlanguage{ngerman}±0.26 & 0.17\selectlanguage{ngerman}±0.78
& 0.13\selectlanguage{ngerman}±0.31\tabularnewline
SPL & X & - & 13.60\selectlanguage{ngerman}±1.26 & - & - & - & -\tabularnewline
& Y & - & 12.70\selectlanguage{ngerman}±1.11 & - & - & - & -\tabularnewline
& Z & - & 8.98\selectlanguage{ngerman}±1.25 & - & - & - & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & 1551.00\selectlanguage{ngerman}±3.38 & - & - & - &
-\tabularnewline
& Y/X & - & 0.28\selectlanguage{ngerman}±1.30 & - & - & - & -\tabularnewline
& percentage & - & 0.27\selectlanguage{ngerman}±0.98 & - & - & - & -\tabularnewline
ML & X & - & - & 6.72\selectlanguage{ngerman}±1.83 & 7.30\selectlanguage{ngerman}±2.91 & - & -\tabularnewline
& Y & - & - & 6.75\selectlanguage{ngerman}±1.72 & 7.08\selectlanguage{ngerman}±2.38 & - & -\tabularnewline
& Z & - & - & 5.00\selectlanguage{ngerman}±0.35 & 5.22\selectlanguage{ngerman}±0.24 & - & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & - & 226.80\selectlanguage{ngerman}±2.31 & 269.79\selectlanguage{ngerman}±1.42 & -
& -\tabularnewline
& Y/X & - & - & 0.79\selectlanguage{ngerman}±0.33 & 0.81\selectlanguage{ngerman}±0.88 & - & -\tabularnewline
& percentage & - & - & 0.04\selectlanguage{ngerman}±0.26 & 0.04\selectlanguage{ngerman}±0.35 & - & -\tabularnewline
TA & X & - & - & 7.39\selectlanguage{ngerman}±2.65 & 7.94\selectlanguage{ngerman}±3.59 & 8.43\selectlanguage{ngerman}±8.22 & -\tabularnewline
& Y & - & - & 7.10\selectlanguage{ngerman}±3.34 & 7.80\selectlanguage{ngerman}±4.07 & 8.35\selectlanguage{ngerman}±6.73 & -\tabularnewline
& Z & - & - & 6.31\selectlanguage{ngerman}±1.85 & 8.64\selectlanguage{ngerman}±1.58 & 6.32\selectlanguage{ngerman}±1.43 & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & - & 331.08\selectlanguage{ngerman}±3.01 & 535.09\selectlanguage{ngerman}±1.38 &
444.87\selectlanguage{ngerman}±2.07 & -\tabularnewline
& Y/X & - & - & 0.96\selectlanguage{ngerman}±0.07 & 0.98\selectlanguage{ngerman}±0.28 & 0.99\selectlanguage{ngerman}±0.72 & -\tabularnewline
& percentage & - & - & 0.06\selectlanguage{ngerman}±0.66 & 0.07\selectlanguage{ngerman}±0.34 & 0.04\selectlanguage{ngerman}±0.58 &
-\tabularnewline
PMC & X & - & 11.86\selectlanguage{ngerman}±2.29 & 12.58\selectlanguage{ngerman}±3.03 & - & - & -\tabularnewline
& Y & - & 11.58\selectlanguage{ngerman}±1.44 & 12.29\selectlanguage{ngerman}±2.89 & - & - & -\tabularnewline
& Z & - & 9.73\selectlanguage{ngerman}±1.36 & 10.73\selectlanguage{ngerman}±2.09 & - & - & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & 1336.31\selectlanguage{ngerman}±5.12 & 1658.95\selectlanguage{ngerman}±1.45 & - &
- & -\tabularnewline
& Y/X & - & 0.98\selectlanguage{ngerman}±0.09 & 0.98\selectlanguage{ngerman}±0.46 & - & - & -\tabularnewline
& percentage & - & 0.31\selectlanguage{ngerman}±0.18 & 0.29\selectlanguage{ngerman}±0.35 & - & - & -\tabularnewline
MP & X & - & - & - & 15.33\selectlanguage{ngerman}±5.78 & 19.60\selectlanguage{ngerman}±4.31 & -\tabularnewline
& Y & - & - & - & 14.76\selectlanguage{ngerman}±5.65 & 20.05\selectlanguage{ngerman}±1.64 & -\tabularnewline
& Z & - & - & - & 12.13\selectlanguage{ngerman}±5.51 & 15.33\selectlanguage{ngerman}±5.97 & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & - & - & 2744.66\selectlanguage{ngerman}±3.07 &
6024.38\selectlanguage{ngerman}±5.31 & -\tabularnewline
& Y/X & - & - & - & 0.96\selectlanguage{ngerman}±0.81 & 1.02\selectlanguage{ngerman}±0.42 & -\tabularnewline
& percentage & - & - & - & 0.33\selectlanguage{ngerman}±0.48 & 0.41\selectlanguage{ngerman}±0.35 & -\tabularnewline
PG & X & - & - & - & - & - & 28.91\selectlanguage{ngerman}±5.34\tabularnewline
& Y & - & - & - & - & - & 30.46\selectlanguage{ngerman}±5.18\tabularnewline
& Z & - & - & - & - & - & 25.15\selectlanguage{ngerman}±6.19\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & - & - & - & - &
22147.05\selectlanguage{ngerman}±0.97\tabularnewline
& Y/X & - & - & - & - & - & 1.05\selectlanguage{ngerman}±0.31\tabularnewline
& percentage & - & - & - & - & - & 0.60\selectlanguage{ngerman}±0.38\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Table 3.} Cell counts in \emph{T. laevis} infected anthers.
X-indicates the cell counts along X-axis, Y-axis indicate cell number
along Y-axis, X*Y is the total cell count per locule and percent mean
percentage of all cells of epidermis (EPI), endothecium (EN), secondary
parietal cell (SPL), middle layer (ML), tapetum (TA), pollen mother cell
(PMC), microspore (MP) and pollen grain (PG). Asterisks* indicate
statistically significant differences. (Student's \emph{t-test}
:\emph{*P} \selectlanguage{english} \selectlanguage{english}[?] 0.05, \emph{**P} \selectlanguage{english} \selectlanguage{english}[?] 0.01). The values without Asterisks*
indicated the statistically non-significant. +- showed the standard
error. -- indicated that cells not appeared.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllllllllll@{}}
\toprule
Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & \textless{} 0-150 &
\textless{} 0-150 & \textless{} 0-150 & 150-400 & 150-400 & 150-400 &
400-700 & 400-700 & 400-700 & 700-1100 & 700-1100 & 700-1100 & 1100-1300
& 1100-1300 & 1100-1300 & 1300-2300 & 1300-2300 &
1300-2300\tabularnewline
& & control & infected & \emph{T-test} & control & infected &
\emph{T-test} & control & infected & \emph{T-test} & control & infected
& \emph{T-test} & control & infected & \emph{T-test} & control &
infected & \emph{T-test}\tabularnewline
EPI & X & 7.00\selectlanguage{ngerman}±0.30 & 9.00\selectlanguage{ngerman}±10.26 & 0.61 & 18.00\selectlanguage{ngerman}±1.41 & 12.50\selectlanguage{ngerman}±6.31 & 0.83
& 19.50\selectlanguage{ngerman}±1.66 & 20.50\selectlanguage{ngerman}±3.94 & 0.88 & 22.00\selectlanguage{ngerman}±1.10 & 26.75\selectlanguage{ngerman}±1.58 & 0.79 &
23.33\selectlanguage{ngerman}±0.18 & 29.17\selectlanguage{ngerman}±7.32 & 0.76 & 24.83\selectlanguage{ngerman}±0.71 & 31.00\selectlanguage{ngerman}±5.56 &
0.53\tabularnewline
& Y & 4.00\selectlanguage{ngerman}±0.40 & 5.00\selectlanguage{ngerman}±2.13 & 0.50 & 16.00\selectlanguage{ngerman}±1.08* & 8.13\selectlanguage{ngerman}±2.75* & 0.05 &
23.16\selectlanguage{ngerman}±2.41* & 15.63\selectlanguage{ngerman}±3.18* & 0.05 & 31.56\selectlanguage{ngerman}±2.20* & 21.25\selectlanguage{ngerman}±4.61* & 0.03 &
34.33\selectlanguage{ngerman}±0.32 & 22.1\selectlanguage{ngerman}±0.89 & 0.06 & 39.18\selectlanguage{ngerman}±5.74* & 25.06\selectlanguage{ngerman}±1.26* &
0.03\tabularnewline
& X*Y & 28.00\selectlanguage{ngerman}±0.30 & 45.00\selectlanguage{ngerman}±0.18 & 0.29 & 288.00\selectlanguage{ngerman}±2.9* & 112.50\selectlanguage{ngerman}±0.78* &
0.02 & 458.87\selectlanguage{ngerman}±5.6** & 340.0\selectlanguage{ngerman}±0.1** & 0.01 & 693.75\selectlanguage{ngerman}±5.79* & 566.75\selectlanguage{ngerman}±0.75* &
0.03 & 801.67\selectlanguage{ngerman}±1.00* & 645.00\selectlanguage{ngerman}±0.29* & 0.03 & 972.82\selectlanguage{ngerman}±4.57* & 778.20\selectlanguage{ngerman}±0.72*
& 0.02\tabularnewline
& percentage & 0.34\selectlanguage{ngerman}±0.15* & 0.21\selectlanguage{ngerman}±0.32* & 0.05 & 0.53\selectlanguage{ngerman}±0.32* & 0.21\selectlanguage{ngerman}±0.22*
& 0.05 & 0.43\selectlanguage{ngerman}±0.10* & 0.32\selectlanguage{ngerman}±0.12* & 0.04 & 0.33\selectlanguage{ngerman}±0.81 & 0.32\selectlanguage{ngerman}±1.32 & 0.48 &
0.32\selectlanguage{ngerman}±0.36 & 0.34\selectlanguage{ngerman}±0.16 & 0.37 & 0.37\selectlanguage{ngerman}±0.33 & 0.41\selectlanguage{ngerman}±0.37 &
0.26\tabularnewline
EN & X & 6.05\selectlanguage{ngerman}±1.08 & 7.00\selectlanguage{ngerman}±4.74 & 0.86 & 7.50\selectlanguage{ngerman}±0.98 & 7.80\selectlanguage{ngerman}±10.39 & 0.75 &
10.03\selectlanguage{ngerman}±0.82 & 8.80\selectlanguage{ngerman}±2.38 & 0.63 & 11.50\selectlanguage{ngerman}±0.71 & 12.83\selectlanguage{ngerman}±3.38 & 0.58 &
12.67\selectlanguage{ngerman}±0.25 & 15.57\selectlanguage{ngerman}±2.13 & 0.55 & 13.84\selectlanguage{ngerman}±0.76 & 15.79\selectlanguage{ngerman}±0.88 &
0.24\tabularnewline
& Y & 9.16\selectlanguage{ngerman}±0.35* & 24.00\selectlanguage{ngerman}±3.30* & 0.04 & 16.00\selectlanguage{ngerman}±2.50* & 35.00\selectlanguage{ngerman}±2.47* & 0.05
& 43.04\selectlanguage{ngerman}±10.07 & 53.50\selectlanguage{ngerman}±6.31 & 0.08 & 93.33\selectlanguage{ngerman}±7.94* & 63.57\selectlanguage{ngerman}±1.58* & 0.05 &
111.33\selectlanguage{ngerman}±0.35* & 63.83\selectlanguage{ngerman}±2.91* & 0.04 & 115.63\selectlanguage{ngerman}±2.32** & 68.80\selectlanguage{ngerman}±7.32** &
0.01\tabularnewline
& X*Y & 54.00\selectlanguage{ngerman}±0.8* & 168.00\selectlanguage{ngerman}±0.1* & 0.03 & 130.50\selectlanguage{ngerman}±2.80 & 281.80\selectlanguage{ngerman}±0.78 &
0.09 & 411.75\selectlanguage{ngerman}±6.45 & 472.30\selectlanguage{ngerman}±0.78 & 0.07 & 1076.33\selectlanguage{ngerman}±1.17* & 819.10\selectlanguage{ngerman}±0.70* &
0.02 & 1411.5\selectlanguage{ngerman}±1.00** & 994.46\selectlanguage{ngerman}±0.73** & 0.01 & 1595.6\selectlanguage{ngerman}±5.06** &
1086.80\selectlanguage{ngerman}±0.1** & 0.01\tabularnewline
& percentage & 0.66\selectlanguage{ngerman}±0.13* & 0.79\selectlanguage{ngerman}±0.42* & 0.04 & 0.24\selectlanguage{ngerman}±0.27* & 0.58\selectlanguage{ngerman}±0.31*
& 0.05 & 0.36\selectlanguage{ngerman}±0.30* & 0.49\selectlanguage{ngerman}±0.72* & 0.05 & 0.48\selectlanguage{ngerman}±0.32 & 0.45\selectlanguage{ngerman}±0.28 & 0.33 &
0.56\selectlanguage{ngerman}±0.53 & 0.52\selectlanguage{ngerman}±0.65 & 0.57 & 0.61\selectlanguage{ngerman}±0.34 & 0.57\selectlanguage{ngerman}±0.39 &
0.40\tabularnewline
SPL & X & - & - & - & 4.85\selectlanguage{ngerman}±0.87 & 5.50\selectlanguage{ngerman}±1.58 & 0.83 & - & - & - & - & - &
- & - & - & - & - & - & -\tabularnewline
& Y & - & - & - & 12.10\selectlanguage{ngerman}±1.59 & 11.00\selectlanguage{ngerman}±3.27 & 0.91 & - & - & - & - & - & -
& - & - & - & - & - & -\tabularnewline
& X*Y & - & - & - & 58.36\selectlanguage{ngerman}±2.02 & 59.50\selectlanguage{ngerman}±0.71 & 0.86 & - & - & - & - & - &
- & - & - & - & - & - & -\tabularnewline
& percentage & - & - & - & 0.14\selectlanguage{ngerman}±0.28 & 0.15\selectlanguage{ngerman}±0.12 & 0.94 & - & - & - & -
& - & - & - & - & - & - & - & -\tabularnewline
ML & X & - & - & - & - & - & - & 5.00\selectlanguage{ngerman}±0.30 & 9.00\selectlanguage{ngerman}±3.11 & 0.25 &
6.75\selectlanguage{ngerman}±0.84* & 12.50\selectlanguage{ngerman}±1.05* & 0.03 & - & - & - & - & - & -\tabularnewline
& Y & - & - & - & - & - & - & 12.50\selectlanguage{ngerman}±0.32 & 8.25\selectlanguage{ngerman}±2.88 & 0.48 &
25.03\selectlanguage{ngerman}±6.75* & 16.10\selectlanguage{ngerman}±3.64* & 0.05 & - & - & - & - & - & -\tabularnewline
& X*Y & - & - & - & - & - & - & 57.00\selectlanguage{ngerman}±0.30* & 77.00\selectlanguage{ngerman}±0.11* & 0.05 &
173.75\selectlanguage{ngerman}±3.67* & 204.75\selectlanguage{ngerman}±0.71* & 0.03 & - & - & - & - & - &
-\tabularnewline
& percentage & - & - & - & - & - & - & 0.06\selectlanguage{ngerman}±0.37 & 0.07\selectlanguage{ngerman}±0.42 & 0.61 &
0.08\selectlanguage{ngerman}±0.51 & 0.11\selectlanguage{ngerman}±0.22 & 0.56 & - & - & - & - & - & -\tabularnewline
TA & X & - & - & - & - & - & - & 5.00\selectlanguage{ngerman}±0.35 & 9.25\selectlanguage{ngerman}±1.45 & 0.53 &
7.33\selectlanguage{ngerman}±1.22* & 11.38\selectlanguage{ngerman}±0.39* & 0.04 & 8.70\selectlanguage{ngerman}±1.15 & 12.75\selectlanguage{ngerman}±4.16 & 0.33 & - & -
& -\tabularnewline
& Y & - & - & - & - & - & - & 11.50\selectlanguage{ngerman}±0.31 & 6.64\selectlanguage{ngerman}±1.89 & 0.09 & 24.00\selectlanguage{ngerman}±1.25
& 16.45\selectlanguage{ngerman}±2.27 & 0.13 & 28.50\selectlanguage{ngerman}±4.51 & 20.45\selectlanguage{ngerman}±1.05 & 0.06 & - & - &
-\tabularnewline
& X*Y & - & - & - & - & - & - & 61.00\selectlanguage{ngerman}±0.50 & 64.65\selectlanguage{ngerman}±0.72 & 0.06 &
179.78\selectlanguage{ngerman}±1.10* & 190.43\selectlanguage{ngerman}±0.18* & 0.05 & 245.75\selectlanguage{ngerman}±3.64* & 260.00\selectlanguage{ngerman}±0.09* & 0.05
& - & - & -\tabularnewline
& percentage & - & - & - & - & - & - & 0.06\selectlanguage{ngerman}±0.50 & 0.06\selectlanguage{ngerman}±0.38 & 0.49 &
0.08\selectlanguage{ngerman}±0.46 & 0.10\selectlanguage{ngerman}±0.12 & 0.36 & 0.10\selectlanguage{ngerman}±0.25 & 0.13\selectlanguage{ngerman}±0.38 & 0.28 & - & - &
-\tabularnewline
PMC & X & - & - & \textbf{-} & 2.00\selectlanguage{ngerman}±0.45 & 2.00\selectlanguage{ngerman}±1.18 & 0.99 & 3.05\selectlanguage{ngerman}±0.22
& 3.00\selectlanguage{ngerman}±0.98 & 0.88 & - & - & - & - & - & - & - & - & -\tabularnewline
& Y & - & - & \textbf{-} & 4.50\selectlanguage{ngerman}±1.79 & 4.00\selectlanguage{ngerman}±0.25 & 0.75 & 9.12\selectlanguage{ngerman}±1.92 &
7.50\selectlanguage{ngerman}±9.42 & 0.43 & - & - & - & - & - & - & - & - & -\tabularnewline
& X*Y & - & - & \textbf{-} & 9.00\selectlanguage{ngerman}±7.12 & 8.00\selectlanguage{ngerman}±0.78 & 0.75 & 30.08\selectlanguage{ngerman}±5.18 &
22.50\selectlanguage{ngerman}±0.28 & 0.07 & - & - & - & - & - & - & - & - & -\tabularnewline
& percentage & - & - & \textbf{-} & 0.03\selectlanguage{ngerman}±0.30 & 0.02\selectlanguage{ngerman}±0.52 & 0.91 &
0.02\selectlanguage{ngerman}±0.61 & 0.02\selectlanguage{ngerman}±0.42 & 0.96 & - & - & - & - & - & - & - & - &
-\tabularnewline
MP & X & - & - & - & - & - & - & - & - & - & 4.03\selectlanguage{ngerman}±1.00 & 4.00\selectlanguage{ngerman}±1.03 &
0.97 & 4.13\selectlanguage{ngerman}±0.16 & 4.00\selectlanguage{ngerman}±1.61 & 0.88 & - & - & -\tabularnewline
& Y & - & - & - & - & - & - & - & - & - & 12.25\selectlanguage{ngerman}±1.15 & 10.75\selectlanguage{ngerman}±5.94 & 0.56
& 13.00\selectlanguage{ngerman}±0.40 & 12.17\selectlanguage{ngerman}±3.93 & 0.86 & - & - & -\tabularnewline
& X*Y & - & - & - & - & - & - & - & - & - & 49.32\selectlanguage{ngerman}±2.31 & 43.00\selectlanguage{ngerman}±0.68 &
0.76 & 53.73\selectlanguage{ngerman}±0.45 & 48.67\selectlanguage{ngerman}±0.21 & 0.88 & - & - & -\tabularnewline
& percentage & - & - & - & - & - & - & - & - & - & 0.02\selectlanguage{ngerman}±0.24 & 0.02\selectlanguage{ngerman}±0.37
& 0.98 & 0.02\selectlanguage{ngerman}±0.39 & 0.02\selectlanguage{ngerman}±0.31 & 0.98 & - & - & -\tabularnewline
PG & X & - & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
4.38\selectlanguage{ngerman}±1.06 & 4.03\selectlanguage{ngerman}±5.18 & 0.72\tabularnewline
& Y & - & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
13.48\selectlanguage{ngerman}±1.14 & 12.70\selectlanguage{ngerman}±1.56 & 0.71\tabularnewline
& X*Y & - & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
59.08\selectlanguage{ngerman}±2.28 & 51.19\selectlanguage{ngerman}±0.15 & 0.11\tabularnewline
& percentage & - & - & - & - & - & - & - & - & - & - & - & - & - & - & -
& 0.02\selectlanguage{ngerman}±0.17 & 0.03\selectlanguage{ngerman}±0.32 & 0.88\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 4.} Cell sizes and volume of anther cells in \emph{T.
laevis} infected anthers. X-axis indicates the cell width, Y-axis cell
length, and Z-axis cell depth of epidermis (EPI), endothecium (EN),
secondary parietal cell (SPL), middle layer (ML), tapetum (TA), pollen
mother cell (PMC), microspore (MP) and pollen grain (PG). Cell volume
was obtained by multiplication of X, Y and Z-axes. Asterisks* indicate
statistically significant differences. (Student's \emph{t-test}
:\emph{*P}  [?] 0.05, \emph{**P}  [?] 0.01). The values without Asterisks*
indicated the statistically non-significant. \selectlanguage{ngerman}± showed the standard
error. -- indicated that cells not appeared.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllllllllll@{}}
\toprule
Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & \textless{} 0-150 &
\textless{} 0-150 & \textless{} 0-150 & 150-400 & 150-400 & 150-400 &
400-700 & 400-700 & 400-700 & 700-1100 & 700-1100 & 700-1100 & 1100-1300
& 1100-1300 & 1100-1300 & 1300-2300 & 1300-2300 &
1300-2300\tabularnewline
& & control & infected & \emph{T-test} & control & infected &
\emph{T-test} & control & infected & \emph{T-test} & control & infected
& \emph{T-test} & control & infected & \emph{T-test} & control &
infected & \emph{T-test}\tabularnewline
EPI & X & 8.00\selectlanguage{ngerman}±1.37 & 5.89\selectlanguage{ngerman}±2.13 & 0.76 & 8.46\selectlanguage{ngerman}±2.33 & 6.73\selectlanguage{ngerman}±3.38 & 0.09 &
9.19\selectlanguage{ngerman}±1.64 & 8.94\selectlanguage{ngerman}±2.75 & 0.38 & 10.29\selectlanguage{ngerman}±2.97 & 11.00\selectlanguage{ngerman}±4.61 & 0.15 &
12.52\selectlanguage{ngerman}±2.45 & 12.79\selectlanguage{ngerman}±0.89 & 0.87 & 14.01\selectlanguage{ngerman}± 4.29 & 13.88\selectlanguage{ngerman}±1.36 &
0.43\tabularnewline
& Y & 16.00\selectlanguage{ngerman}±4.13 & 18.16\selectlanguage{ngerman}±6.31 & 0.83 & 16.75\selectlanguage{ngerman}±12.45 & 19.31\selectlanguage{ngerman}±10.79 & 0.51
& 21.25\selectlanguage{ngerman}±3.22 & 25.41\selectlanguage{ngerman}±3.94 & 0.62 & 34.63\selectlanguage{ngerman}±11.32 & 35.13\selectlanguage{ngerman}±7.34 & 0.71 &
47.10\selectlanguage{ngerman}±8.15* & 59.17\selectlanguage{ngerman}±2.45* & 0.05 & 51.08\selectlanguage{ngerman}±9.80* & 76.42\selectlanguage{ngerman}±5.56* &
0.05\tabularnewline
& Z & 6.67\selectlanguage{ngerman}±3.01 & 4.58\selectlanguage{ngerman}±0.49 & 0.87 & 6.78\selectlanguage{ngerman}±1.69 & 4.95\selectlanguage{ngerman}±2.44 & 0.39 &
7.07\selectlanguage{ngerman}±0.33 & 6.27\selectlanguage{ngerman}±0.31 & 0.85 & 7.79\selectlanguage{ngerman}±1.39 & 7.31\selectlanguage{ngerman}±0.88 & 0.63 & 9.71\selectlanguage{ngerman}±3.54
& 7.51\selectlanguage{ngerman}±0.02 & 0.07 & 11.98\selectlanguage{ngerman}±2.13 & 8.63\selectlanguage{ngerman}±6.08 & 0.06\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & 853.76\selectlanguage{ngerman}±2.37* & 489.89\selectlanguage{ngerman}±0.53* & 0.03 &
1074.3\selectlanguage{ngerman}±1.13* & 643.28\selectlanguage{ngerman}±0.27* & 0.04 & 1395.68\selectlanguage{ngerman}±1.3* & 1424.33\selectlanguage{ngerman}±0.3* & 0.05
& 2775.9\selectlanguage{ngerman}±1.65* & 2824.80\selectlanguage{ngerman}±0.3* & 0.05 & 5725.91\selectlanguage{ngerman}±1.38 & 5683.45\selectlanguage{ngerman}±0.26 &
0.83 & 8573.3\selectlanguage{ngerman}±2.26* & 9153.92\selectlanguage{ngerman}±0.16* & 0.04\tabularnewline
& Y/X & 2.00\selectlanguage{ngerman}±0.36* & 3.08\selectlanguage{ngerman}±0.92* & 0.05 & 1.98\selectlanguage{ngerman}±0.12 & 2.87\selectlanguage{ngerman}±0.16 & 0.86 &
2.30\selectlanguage{ngerman}±0.39 & 2.84\selectlanguage{ngerman}±0.36 & 0.88 & 3.33\selectlanguage{ngerman}±0.41 & 3.19\selectlanguage{ngerman}±0.66 & 0.92 & 3.77\selectlanguage{ngerman}±0.08
& 4.63\selectlanguage{ngerman}±0.24 & 0.81 & 3.64\selectlanguage{ngerman}±0.95* & 5.51\selectlanguage{ngerman}±0.37* & 0.03\tabularnewline
& percentage & 0.51\selectlanguage{ngerman}±0.11* & 0.28\selectlanguage{ngerman}±0.25* & 0.04 & 0.24\selectlanguage{ngerman}±0.30* & 0.12\selectlanguage{ngerman}±0.30*
& 0.05 & 0.25\selectlanguage{ngerman}±0.52 & 0.20\selectlanguage{ngerman}±0.43 & 0.63 & 0.33\selectlanguage{ngerman}±0.37 & 0.28\selectlanguage{ngerman}±0.05 & 0.54 &
0.39\selectlanguage{ngerman}±0.10 & 0.34\selectlanguage{ngerman}±0.16 & 0.71 & 0.27\selectlanguage{ngerman}±0.37 & 0.24\selectlanguage{ngerman}±0.29 &
0.66\tabularnewline
EN & X & 10.21\selectlanguage{ngerman}±1.78 & 13.51\selectlanguage{ngerman}±4.74 & 0.91 & 12.67\selectlanguage{ngerman}±3.69 & 14.37\selectlanguage{ngerman}±2.38 & 0.82
& 18.65\selectlanguage{ngerman}±5.36 & 17.71\selectlanguage{ngerman}±10.39 & 0.54 & 23.35\selectlanguage{ngerman}±9.75 & 25.11\selectlanguage{ngerman}±1.38 & 0.07 &
27.91\selectlanguage{ngerman}±8.42* & 33.29\selectlanguage{ngerman}±3.22* & 0.05 & 30.82\selectlanguage{ngerman}±11.51* & 38.72\selectlanguage{ngerman}±0.99* &
0.04\tabularnewline
& Y & 8.03\selectlanguage{ngerman}±1.89 & 5.32\selectlanguage{ngerman}±2.27 & 0.64 & 8.75\selectlanguage{ngerman}±1.69 & 5.85\selectlanguage{ngerman}±3.30 & 0.07 &
9.75\selectlanguage{ngerman}±1.52 & 8.17\selectlanguage{ngerman}±2.47 & 0.92 & 10.40\selectlanguage{ngerman}±2.15 & 10.59\selectlanguage{ngerman}±8.25 & 0.37 &
10.77\selectlanguage{ngerman}±1.39 & 11.90\selectlanguage{ngerman}±0.96 & 0.18 & 13.54\selectlanguage{ngerman}±4.81 & 13.50\selectlanguage{ngerman}±7.31 &
0.42\tabularnewline
& Z & 5.22\selectlanguage{ngerman}±0.26 & 4.68\selectlanguage{ngerman}±0.51 & 0.83 & 5.47\selectlanguage{ngerman}±1.21 & 4.87\selectlanguage{ngerman}±2.29 & 0.73 &
6.58\selectlanguage{ngerman}±0.27 & 5.75\selectlanguage{ngerman}±0.90 & 0.29 & 7.55\selectlanguage{ngerman}±0.51 & 6.97\selectlanguage{ngerman}±1.53 & 0.86 & 8.09\selectlanguage{ngerman}±1.05
& 7.57\selectlanguage{ngerman}±0.35 & 0.16 & 9.23\selectlanguage{ngerman}±1.67 & 8.89\selectlanguage{ngerman}±1.49 & 0.55\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & 427.97\selectlanguage{ngerman}±1.35* & 336.37\selectlanguage{ngerman}±0.86* & 0.05 &
606.42\selectlanguage{ngerman}±0.57* & 409.39\selectlanguage{ngerman}±0.52* & 0.04 & 1196.5\selectlanguage{ngerman}±1.75* & 831.97\selectlanguage{ngerman}±0.81* & 0.03
& 1833.44\selectlanguage{ngerman}±1.46 & 1853.43\selectlanguage{ngerman}±0.19 & 0.29 & 2431.78\selectlanguage{ngerman}±2.3* & 2998.86\selectlanguage{ngerman}±0.15* &
0.02 & 3851.7\selectlanguage{ngerman}±3.4** & 4647.0\selectlanguage{ngerman}±0.06** & 0.01\tabularnewline
& Y/X & 0.79\selectlanguage{ngerman}±0.92* & 0.39\selectlanguage{ngerman}±0.32* & 0.05 & 0.69\selectlanguage{ngerman}±0.35 & 0.41\selectlanguage{ngerman}±1.42 & 0.04 &
0.52\selectlanguage{ngerman}±0.13 & 0.46\selectlanguage{ngerman}±0.21 & 0.63 & 0.45\selectlanguage{ngerman}±0.47 & 0.42\selectlanguage{ngerman}±0.16 & 0.45 & 0.39\selectlanguage{ngerman}±0.79
& 0.36\selectlanguage{ngerman}±0.76 & 0.84 & 0.44\selectlanguage{ngerman}±0.34 & 0.35\selectlanguage{ngerman}±0.21 & 0.26\tabularnewline
& percentage & 0.49\selectlanguage{ngerman}±0.98 & 0.72\selectlanguage{ngerman}±0.25 & 0.19 & 0.14\selectlanguage{ngerman}±0.82 & 0.80\selectlanguage{ngerman}±0.30 &
0.09 & 0.22\selectlanguage{ngerman}±0.35* & 0.12\selectlanguage{ngerman}±0.03* & 0.03 & 0.23\selectlanguage{ngerman}±0.26 & 0.19\selectlanguage{ngerman}±0.39 & 0.38 &
0.17\selectlanguage{ngerman}±0.26 & 0.21\selectlanguage{ngerman}±0.58 & 0.43 & 0.13\selectlanguage{ngerman}±0.31 & 0.18\selectlanguage{ngerman}±0.46 &
0.61\tabularnewline
SPL & X & & - & - & 13.60\selectlanguage{ngerman}±1.26 & 12.35\selectlanguage{ngerman}±0.39 & 0.56 & - & - & - & - & - &
- & - & - & - & - & - & -\tabularnewline
& Y & & - & - & 12.70\selectlanguage{ngerman}±1.11 & 10.87\selectlanguage{ngerman}±1.93 & 0.49 & - & - & - & - & - & - &
- & - & - & - & - & -\tabularnewline
& Z & & - & - & 8.98\selectlanguage{ngerman}±1.25 & 8.42\selectlanguage{ngerman}±1.67 & 0.81 & - & - & - & - & - & - & -
& - & - & - & - & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & & - & - & 1551.00\selectlanguage{ngerman}±3.4* & 1130.34\selectlanguage{ngerman}±0.2*
& 0.05 & - & - & - & - & - & - & - & - & - & - & - & -\tabularnewline
& Y/X & & - & - & 0.28\selectlanguage{ngerman}±1.30* & 0.88\selectlanguage{ngerman}±0.92* & 0.05 & - & - & - & - & - & -
& - & - & - & - & - & -\tabularnewline
& percentage & & - & - & 0.27\selectlanguage{ngerman}±0.98 & 0.22\selectlanguage{ngerman}±0.16 & 0.32 & - & - & - & - &
- & - & - & - & - & - & - & -\tabularnewline
ML & X & & - & - & - & - & - & 6.72\selectlanguage{ngerman}±1.83 & 8.35\selectlanguage{ngerman}±1.05 & 0.61 & 7.30\selectlanguage{ngerman}±2.91
& 10.20\selectlanguage{ngerman}±2.13 & 0.69 & - & - & - & - & - & -\tabularnewline
& Y & & - & - & - & - & - & 6.75\selectlanguage{ngerman}±1.72 & 4.49\selectlanguage{ngerman}±1.59 & 0.39 & 7.08\selectlanguage{ngerman}±2.38 &
4.65\selectlanguage{ngerman}±3.64 & 0.35 & - & - & - & - & - & -\tabularnewline
& Z & & - & - & - & - & - & 5.00\selectlanguage{ngerman}±0.35 & 4.82\selectlanguage{ngerman}±2.33 & 0.47 & 5.22\selectlanguage{ngerman}±0.24 &
5.58\selectlanguage{ngerman}±0.58 & 0.28 & - & - & - & - & - & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & & - & - & - & - & - & 226.80\selectlanguage{ngerman}±2.31* &
180.71\selectlanguage{ngerman}±0.96* & 0.05 & 269.79\selectlanguage{ngerman}±1.42 & 264.66\selectlanguage{ngerman}±0.23 & 0.67 & - & - & - & - &
- & -\tabularnewline
& Y/X & & - & - & - & - & - & 0.79\selectlanguage{ngerman}±0.33* & 0.54\selectlanguage{ngerman}±0.13* & 0.05 & 0.81\selectlanguage{ngerman}±0.88
& 0.46\selectlanguage{ngerman}±0.30 & 0.13 & - & - & - & - & - & -\tabularnewline
& percentage & & - & - & - & - & - & 0.04\selectlanguage{ngerman}±0.26 & 0.25\selectlanguage{ngerman}±0.27 & 0.25 &
0.04\selectlanguage{ngerman}±0.35 & 0.25\selectlanguage{ngerman}±0.16 & 0.31 & - & - & - & - & & -\tabularnewline
TA & X & & - & - & - & - & - & 7.39\selectlanguage{ngerman}±2.65 & 6.96\selectlanguage{ngerman}±1.45 & 0.23 & 7.94\selectlanguage{ngerman}±3.59
& 7.17\selectlanguage{ngerman}±0.39 & 0.90 & 8.43\selectlanguage{ngerman}±8.22 & 7.29\selectlanguage{ngerman}±3.31 & 0.16 & - & - &
-\tabularnewline
& Y & & - & - & - & - & - & 7.10\selectlanguage{ngerman}±3.34 & 8.71\selectlanguage{ngerman}±1.89 & 0.71 & 7.80\selectlanguage{ngerman}±4.07 &
11.71\selectlanguage{ngerman}±2.27 & 0.86 & 8.35\selectlanguage{ngerman}±6.73 & 8.77\selectlanguage{ngerman}±3.30 & 0.35 & - & - &
-\tabularnewline
& Z & & - & - & - & - & - & 6.31\selectlanguage{ngerman}±1.85 & 4.37\selectlanguage{ngerman}±2.63 & 0.62 & 8.64\selectlanguage{ngerman}±1.58 &
5.05\selectlanguage{ngerman}±0.37 & 0.44 & 6.32\selectlanguage{ngerman}±1.43 & 5.59\selectlanguage{ngerman}±1.52 & 0.37 & - & - &
-\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & & - & - & - & - & - & 331.08\selectlanguage{ngerman}±3.01* &
264.92\selectlanguage{ngerman}±0.23* & 0.05 & 535.09\selectlanguage{ngerman}±1.38* & 424.00\selectlanguage{ngerman}±0.21* & 0.04 & 444.87\selectlanguage{ngerman}±2.07*
& 357.39\selectlanguage{ngerman}±0.62* & 0.04 & - & - & -\tabularnewline
& Y/X & & - & - & - & - & - & 0.96\selectlanguage{ngerman}±0.07 & 1.25\selectlanguage{ngerman}±0.31 & 0.82 & 0.98\selectlanguage{ngerman}±0.28 &
1.63\selectlanguage{ngerman}±0.86 & 0.96 & 0.99\selectlanguage{ngerman}±0.72 & 1.20\selectlanguage{ngerman}±0.91 & 0.81 & - & - &
-\tabularnewline
& percentage & & - & - & - & - & - & 0.06\selectlanguage{ngerman}±0.66 & 0.37\selectlanguage{ngerman}±0.27 & 0.51 &
0.07\selectlanguage{ngerman}±0.34 & 0.44\selectlanguage{ngerman}±0.72 & 0.36 & 0.04\selectlanguage{ngerman}±0.58 & 0.27\selectlanguage{ngerman}±0.36 & 0.43 & - & - &
-\tabularnewline
PMC & X & & - & - & 11.86\selectlanguage{ngerman}±2.29 & 15.06\selectlanguage{ngerman}±1.18 & 0.11 & 12.58\selectlanguage{ngerman}±3.03 &
15.84\selectlanguage{ngerman}±2.39 & 0.18 & - & - & - & - & - & - & - & - & -\tabularnewline
& Y & & - & - & 11.58\selectlanguage{ngerman}±1.44 & 14.92\selectlanguage{ngerman}±0.25 & 0.19 & 12.29\selectlanguage{ngerman}±2.89 & 15.78\selectlanguage{ngerman}±6.31
& 0.23 & - & - & - & - & - & - & - & - & -\tabularnewline
& Z & & - & - & 9.73\selectlanguage{ngerman}±1.36 & 12.36\selectlanguage{ngerman}±0.06 & 0.28 & 10.73\selectlanguage{ngerman}±2.09 & 14.71\selectlanguage{ngerman}±2.35
& 0.56 & - & - & - & - & - & - & - & - & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & & - & - & 1336.3\selectlanguage{ngerman}±5.1** & 2777.2\selectlanguage{ngerman}±0.3**
& 0.01 & 1658.9\selectlanguage{ngerman}±1.4** & 3676.8\selectlanguage{ngerman}±0.2** & 0.01 & - & - & - & - & - & - & -
& - & -\tabularnewline
& Y/X & & - & - & 0.98\selectlanguage{ngerman}±0.09 & 0.99\selectlanguage{ngerman}±0.86 & 0.97 & 0.98\selectlanguage{ngerman}±0.46 & 1.00\selectlanguage{ngerman}±0.18 &
0.90 & - & - & - & - & - & - & - & - & -\tabularnewline
& percentage & & - & - & 0.31\selectlanguage{ngerman}±0.18* & 0.55\selectlanguage{ngerman}±0.27* & 0.03 & 0.29\selectlanguage{ngerman}±0.35* &
0.51\selectlanguage{ngerman}±0.15* & 0.04 & - & - & - & - & - & - & - & - & -\tabularnewline
MP & X & & - & - & - & - & - & - & - & - & 15.33\selectlanguage{ngerman}±5.78 & 16.85\selectlanguage{ngerman}±1.03 &
0.69 & 19.60\selectlanguage{ngerman}±4.31 & 18.32\selectlanguage{ngerman}±1.61 & 0.74 & - & - & -\tabularnewline
& Y & & - & - & - & - & - & - & - & - & 14.76\selectlanguage{ngerman}±5.65 & 16.70\selectlanguage{ngerman}±10.05 & 0.76
& 20.05\selectlanguage{ngerman}±1.64 & 18.53\selectlanguage{ngerman}±3.93 & 0.91 & - & - & -\tabularnewline
& Z & & - & - & - & - & - & - & - & - & 12.13\selectlanguage{ngerman}±5.51 & 16.13\selectlanguage{ngerman}±1.43 & 0.88 &
15.33\selectlanguage{ngerman}±5.97 & 17.65\selectlanguage{ngerman}±0.93 & 0.53 & - & - & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & & - & - & - & - & - & - & - & - &
2744.6\selectlanguage{ngerman}±3.07* & 4538.9\selectlanguage{ngerman}±0.24* & 0.05 & 6024.38\selectlanguage{ngerman}±5.31 & 5991.64\selectlanguage{ngerman}±0.06 & 0.19
& - & - & -\tabularnewline
& Y/X & & - & - & - & - & - & - & - & - & 0.96\selectlanguage{ngerman}±0.81 & 0.99\selectlanguage{ngerman}±0.71 & 0.88 &
1.02\selectlanguage{ngerman}±0.42 & 1.01\selectlanguage{ngerman}±0.29 & 0.97 & - & - & -\tabularnewline
& percentage & & - & - & - & - & - & - & - & - & 0.33\selectlanguage{ngerman}±0.48 & 0.43\selectlanguage{ngerman}±0.32 &
0.19 & 0.41\selectlanguage{ngerman}±0.35 & 0.39\selectlanguage{ngerman}±0.43 & 0.24 & - & - & -\tabularnewline
PG & X & & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
28.91\selectlanguage{ngerman}±5.34 & 24.79\selectlanguage{ngerman}±5.18 & 0.18\tabularnewline
& Y & & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
30.46\selectlanguage{ngerman}±5.18* & 25.87\selectlanguage{ngerman}±1.58* & 0.05\tabularnewline
& Z & & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
25.15\selectlanguage{ngerman}±6.19 & 24.55\selectlanguage{ngerman}±1.51 & 0.43\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & & - & - & - & - & - & - & - & - & - &
- & - & - & - & - & 22147.\selectlanguage{ngerman}±0.9** & 15744.3\selectlanguage{ngerman}±0.2** & 0.01\tabularnewline
& Y/X & & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
1.05\selectlanguage{ngerman}±0.31 & 1.04\selectlanguage{ngerman}±0.29 & 0.92\tabularnewline
& percentage & & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
0.60\selectlanguage{ngerman}±0.38 & 0.58\selectlanguage{ngerman}±0.14 & 0.41\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 5.} Cell counts in \emph{controversa} infected anthers.
X-indicates the cell counts along X-axis, Y-axis indicate cell number
along Y-axis, X*Y is the total cell count per locule and percent mean
percentage of all cells of epidermis (EPI), endothecium (EN), secondary
parietal cell (SPL), middle layer (ML), tapetum (TA), pollen mother cell
(PMC), microspore (MP) and pollen grain (PG). Asterisks* indicate
statistically significant differences. (Student's \emph{t-test}
:\emph{*P}  [?] 0.05, \emph{**P}  [?] 0.01). The values without Asterisks*
indicated the statistically non-significant. \selectlanguage{ngerman}± showed standard error. --
indicated that cells not appeared.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllllllllll@{}}
\toprule
Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & \textless{} 0-150 &
\textless{} 0-150 & \textless{} 0-150 & 150-400 & 150-400 & 150-400 &
400-700 & 400-700 & 400-700 & 700-1100 & 700-1100 & 700-1100 & 1100-1300
& 1100-1300 & 1100-1300 & 1300-2300 & 1300-2300 &
1300-2300\tabularnewline
& & control & infected & \emph{T-test} & control & infected &
\emph{T-test} & control & infected & \emph{T-test} & control & infected
& \emph{T-test} & control & infected & \emph{T-test} & control &
infected & \emph{T-test}\tabularnewline
EPI & X & 7.00\selectlanguage{ngerman}±0.30* & 15.00\selectlanguage{ngerman}±2.08* & 0.05 & 18.00\selectlanguage{ngerman}±1.41 & 17.50\selectlanguage{ngerman}±1.37 &
0.90 & 19.50\selectlanguage{ngerman}±1.66 & 21.50\selectlanguage{ngerman}±2.97 & 0.93 & 22.00\selectlanguage{ngerman}±1.10 & 24.33\selectlanguage{ngerman}±4.29 & 0.89 &
23.33\selectlanguage{ngerman}±0.18 & 26.00\selectlanguage{ngerman}±2.30 & 0.89 & 24.83\selectlanguage{ngerman}±0.71 & 26.30\selectlanguage{ngerman}±5.78 &
0.96\tabularnewline
& Y & 4.00\selectlanguage{ngerman}±0.40* & 13.80\selectlanguage{ngerman}±2.88* & 0.03 & 16.00\selectlanguage{ngerman}±1.08 & 15.10\selectlanguage{ngerman}±2.95 & 0.89 &
23.16\selectlanguage{ngerman}±2.41 & 21.09\selectlanguage{ngerman}±2.77 & 0.80 & 31.56\selectlanguage{ngerman}±2.20 & 38.69\selectlanguage{ngerman}±2.44 & 0.39 &
34.33\selectlanguage{ngerman}±0.32* & 46.33\selectlanguage{ngerman}±1.38* & 0.04 & 39.18\selectlanguage{ngerman}±5.74* & 50.00\selectlanguage{ngerman}±0.61* &
0.05\tabularnewline
& X*Y & 28.00\selectlanguage{ngerman}±0.30** & 207.0\selectlanguage{ngerman}±0.27** & 0.01 & 288.00\selectlanguage{ngerman}±2.86 & 267.50\selectlanguage{ngerman}±0.35 &
0.46 & 458.87\selectlanguage{ngerman}±5.64 & 460.47\selectlanguage{ngerman}±0.61 & 0.75 & 693.75\selectlanguage{ngerman}±5.79* & 966.24\selectlanguage{ngerman}±0.92* &
0.04 & 801.67\selectlanguage{ngerman}±1.00* & 1204.67\selectlanguage{ngerman}±0.26* & 0.03 & 972.82\selectlanguage{ngerman}±4.57* & 1315.5\selectlanguage{ngerman}±0.7*
& 0.02\tabularnewline
& percentage & 0.34\selectlanguage{ngerman}±0.15 & 0.78\selectlanguage{ngerman}±0.46 & 0.52 & 0.53\selectlanguage{ngerman}±0.32 & 0.53\selectlanguage{ngerman}±0.85 &
0.61 & 0.43\selectlanguage{ngerman}±0.10 & 0.42\selectlanguage{ngerman}±0.35 & 0.55 & 0.33\selectlanguage{ngerman}±0.81 & 0.38\selectlanguage{ngerman}±0.25 & 0.43 &
0.32\selectlanguage{ngerman}±0.36 & 0.40\selectlanguage{ngerman}±0.35 & 0.29 & 0.37\selectlanguage{ngerman}±0.33 & 0.42\selectlanguage{ngerman}±0.25 &
0.17\tabularnewline
EN & X & 6.05\selectlanguage{ngerman}±1.08* & 13.00\selectlanguage{ngerman}±1.81* & 0.05 & 7.50\selectlanguage{ngerman}±0.98* & 13.50\selectlanguage{ngerman}±1.79* &
0.05 & 10.03\selectlanguage{ngerman}±0.82 & 15.00\selectlanguage{ngerman}±9.57 & 0.77 & 11.50\selectlanguage{ngerman}±0.71* & 17.25\selectlanguage{ngerman}±8.42* & 0.05
& 12.67\selectlanguage{ngerman}±0.25 & 18.50\selectlanguage{ngerman}±3.03 & 0.71 & 13.84\selectlanguage{ngerman}±0.76* & 20.25\selectlanguage{ngerman}±5.78* &
0.04\tabularnewline
& Y & 9.16\selectlanguage{ngerman}±0.35 & 4.56\selectlanguage{ngerman}±0.40 & 0.38 & 16.00\selectlanguage{ngerman}±2.50 & 10.38\selectlanguage{ngerman}±0.44 & 0.53 &
43.04\selectlanguage{ngerman}±10.07* & 29.30\selectlanguage{ngerman}±1.59* & 0.05 & 93.33\selectlanguage{ngerman}±7.94* & 69.65\selectlanguage{ngerman}±3.61* & 0.04 &
111.33\selectlanguage{ngerman}±0.35* & 84.5\selectlanguage{ngerman}±0.97* & 0.02 & 115.63\selectlanguage{ngerman}±2.32* & 86.82\selectlanguage{ngerman}±3.28* &
0.02\tabularnewline
& X*Y & 54.00\selectlanguage{ngerman}±0.78 & 59.40\selectlanguage{ngerman}±0.13 & 0.61 & 130.50\selectlanguage{ngerman}±2.80 & 143.55\selectlanguage{ngerman}±0.37 &
0.08 & 411.75\selectlanguage{ngerman}±6.45* & 452.65\selectlanguage{ngerman}±0.53* & 0.05 & 1076.33\selectlanguage{ngerman}±1.17* &
1184.03\selectlanguage{ngerman}±0.34* & 0.05 & 1411.50\selectlanguage{ngerman}±1.00* & 1551.93\selectlanguage{ngerman}±0.39* & 0.05 &
1595.64\selectlanguage{ngerman}±5.06* & 1755.4\selectlanguage{ngerman}±0.7* & 0.04\tabularnewline
& percentage & 0.66\selectlanguage{ngerman}±0.13 & 0.22\selectlanguage{ngerman}±0.23 & 0.25 & 0.24\selectlanguage{ngerman}±0.27 & 0.24\selectlanguage{ngerman}±0.23 &
0.31 & 0.36\selectlanguage{ngerman}±0.30 & 0.38\selectlanguage{ngerman}±0.45 & 0.18 & 0.48\selectlanguage{ngerman}±0.32 & 0.46\selectlanguage{ngerman}±0.75 & 0.11 &
0.56\selectlanguage{ngerman}±0.53 & 0.51\selectlanguage{ngerman}±0.61 & 0.37 & 0.61\selectlanguage{ngerman}±0.34 & 0.56\selectlanguage{ngerman}±0.40 &
0.26\tabularnewline
SPL & X & - & - & - & 4.85\selectlanguage{ngerman}±0.87 & 4.94\selectlanguage{ngerman}±0.89 & 0.83 & - & - & - & - & - &
- & - & - & - & - & - & -\tabularnewline
& Y & - & - & - & 12.10\selectlanguage{ngerman}±1.59 & 12.13\selectlanguage{ngerman}±0.76 & 0.94 & - & - & - & - & - & -
& - & - & - & - & - & -\tabularnewline
& X*Y & - & - & - & 58.36\selectlanguage{ngerman}±2.02 & 59.65\selectlanguage{ngerman}±0.72 & 0.76 & - & - & - & - & - &
- & - & - & - & - & - & -\tabularnewline
& percentage & - & - & - & 0.14\selectlanguage{ngerman}±0.28 & 0.06\selectlanguage{ngerman}±0.35 & 0.14 & - & - & - & -
& - & - & - & - & - & - & - & -\tabularnewline
ML & X & - & - & - & - & - & - & 5.00\selectlanguage{ngerman}±0.30 & 8.50\selectlanguage{ngerman}±0.88 & 0.09 &
6.75\selectlanguage{ngerman}±0.84* & 10.25\selectlanguage{ngerman}±1.56* & 0.05 & - & - & - & - & - & -\tabularnewline
& Y & - & - & - & - & - & - & 12.50\selectlanguage{ngerman}±0.32 & 11.12\selectlanguage{ngerman}±0.82 & 0.46 &
25.03\selectlanguage{ngerman}±6.75 & 20.05\selectlanguage{ngerman}±0.68 & 0.37 & - & - & - & - & - & -\tabularnewline
& X*Y & - & - & - & - & - & - & 57.00\selectlanguage{ngerman}±0.30* & 95.40\selectlanguage{ngerman}±0.77* & 0.03 &
173.75\selectlanguage{ngerman}±3.67* & 206.48\selectlanguage{ngerman}±0.73* & 0.02 & - & - & - & - & - &
-\tabularnewline
& percentage & - & - & - & - & - & - & 0.06\selectlanguage{ngerman}±0.37 & 0.09\selectlanguage{ngerman}±0.65 & 0.29 &
0.08\selectlanguage{ngerman}±0.51 & 0.08\selectlanguage{ngerman}±0.15 & 0.96 & - & - & - & - & - & -\tabularnewline
TA & X & - & - & - & - & - & - & 5.00\selectlanguage{ngerman}±0.35 & 6.50\selectlanguage{ngerman}±1.47 & 0.41 &
7.33\selectlanguage{ngerman}±1.22 & 8.75\selectlanguage{ngerman}±3.03 & 0.44 & 8.70\selectlanguage{ngerman}±1.15 & 10.00\selectlanguage{ngerman}±5.78 & 0.60 & - & - &
-\tabularnewline
& Y & - & - & - & - & - & - & 11.50\selectlanguage{ngerman}±0.31 & 10.35\selectlanguage{ngerman}±0.74 & 0.52 &
24.00\selectlanguage{ngerman}±1.25 & 21.76\selectlanguage{ngerman}±0.96 & 0.13 & 28.50\selectlanguage{ngerman}±4.51 & 26.70\selectlanguage{ngerman}±0.93 & 0.26 & - & -
& -\tabularnewline
& X*Y & - & - & - & - & - & - & 61.00\selectlanguage{ngerman}±0.50* & 69.30\selectlanguage{ngerman}±0.91* & 0.05 &
179.78\selectlanguage{ngerman}±1.10* & 194.30\selectlanguage{ngerman}±0.26* & 0.05 & 245.75\selectlanguage{ngerman}±3.64* & 267.20\selectlanguage{ngerman}±0.64* & 0.05
& - & - & -\tabularnewline
& percentage & - & - & - & - & - & - & 0.06\selectlanguage{ngerman}±0.50 & 0.06\selectlanguage{ngerman}±0.25 & 0.98 &
0.08\selectlanguage{ngerman}±0.46 & 0.07\selectlanguage{ngerman}±0.37 & 0.81 & 0.10\selectlanguage{ngerman}±0.25 & 0.09\selectlanguage{ngerman}±0.42 & 0.80 & - & - &
-\tabularnewline
PMC & X & - & - & - & 2.00\selectlanguage{ngerman}±0.45 & 2.25\selectlanguage{ngerman}±0.82 & 1.00 & 3.05\selectlanguage{ngerman}±0.22 &
3.25\selectlanguage{ngerman}±1.58 & 0.67 & - & - & - & - & - & - & - & - & -\tabularnewline
& Y & - & - & - & 4.50\selectlanguage{ngerman}±1.79 & 3.50\selectlanguage{ngerman}±0.87 & 0.70 & 9.12\selectlanguage{ngerman}±1.92* & 6.75\selectlanguage{ngerman}±0.89*
& 0.05 & - & - & - & - & - & - & - & - & -\tabularnewline
& X*Y & - & - & - & 9.00\selectlanguage{ngerman}±7.12 & 8.00\selectlanguage{ngerman}±0.78 & 0.70 & 30.08\selectlanguage{ngerman}±5.18* &
24.00\selectlanguage{ngerman}±0.62* & 0.05 & - & - & - & - & - & - & - & - & -\tabularnewline
& percentage & - & - & - & 0.03\selectlanguage{ngerman}±0.30 & 0.02\selectlanguage{ngerman}±0.44 & 0.93 & 0.02\selectlanguage{ngerman}±0.61 &
0.02\selectlanguage{ngerman}±0.55 & 0.99 & - & - & - & - & - & - & - & - & -\tabularnewline
MP & X & - & - & - & - & - & - & - & - & - & 4.03\selectlanguage{ngerman}±1.00 & 4.50\selectlanguage{ngerman}±4.31 &
0.89 & 4.13\selectlanguage{ngerman}±0.16 & 5.00\selectlanguage{ngerman}±1.34 & 0.81 & - & - & -\tabularnewline
& Y & - & - & - & - & - & - & - & - & - & 12.25\selectlanguage{ngerman}±1.15 & 9.68\selectlanguage{ngerman}±1.64 & 0.92
& 13.00\selectlanguage{ngerman}±0.40 & 10.07\selectlanguage{ngerman}±0.86 & 0.95 & - & - & -\tabularnewline
& X*Y & - & - & - & - & - & - & - & - & - & 49.32\selectlanguage{ngerman}±2.31 & 43.50\selectlanguage{ngerman}±0.35 &
0.92 & 53.73\selectlanguage{ngerman}±0.45 & 50.33\selectlanguage{ngerman}±0.15 & 0.84 & - & - & -\tabularnewline
& percentage & - & - & - & - & - & - & - & - & - & 0.02\selectlanguage{ngerman}±0.24 & 0.02\selectlanguage{ngerman}±0.31
& 0.98 & 0.02\selectlanguage{ngerman}±0.39 & 0.02\selectlanguage{ngerman}±0.25 & 0.98 & - & - & -\tabularnewline
PG & X & - & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
4.38\selectlanguage{ngerman}±1.06 & 5.20\selectlanguage{ngerman}±2.39 & 0.93\tabularnewline
& Y & - & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
13.48\selectlanguage{ngerman}±1.14 & 10.01\selectlanguage{ngerman}±2.69 & 0.92\tabularnewline
& X*Y & - & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
59.08\selectlanguage{ngerman}±2.28* & 52.20\selectlanguage{ngerman}±0.63* & 0.05\tabularnewline
& percentage & - & - & - & - & - & - & - & - & - & - & - & - & - & - & -
& 0.02\selectlanguage{ngerman}±0.17 & 0.02\selectlanguage{ngerman}±0.45 & 0.98\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 6.} Cell sizes and volume of anther cells in \emph{T.
controversa} infected anthers. X-axis indicates the cell width, Y-axis
cell length, and Z-axis cell depth of epidermis (EPI), endothecium (EN),
secondary parietal cell (SPL), middle layer (ML), tapetum (TA), pollen
mother cell (PMC), microspore (MP) and pollen grain (PG). Cell volume
obtained by multiplication of X, Y and Z-axes. Asterisks* indicate
statistically significant differences. (Student's \emph{t-test}
:\emph{*P}  [?] 0.05, \emph{**P}  [?] 0.01). The values without Asterisks*
indicated the statistically non-significant. \selectlanguage{ngerman}± showed the standard
error. -- indicated that cells not appeared.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllllllllllllll@{}}
\toprule
Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & Anther length (\selectlanguage{greek}μ\selectlanguage{english}m) & \textless{} 0-150 &
\textless{} 0-150 & \textless{} 0-150 & 150-400 & 150-400 & 150-400 &
400-700 & 400-700 & 400-700 & 700-1100 & 700-1100 & 700-1100 & 1100-1300
& 1100-1300 & 1100-1300 & 1300-2300 & 1300-2300 &
1300-2300\tabularnewline
& & control & infected & \emph{T-test} & control & infected &
\emph{T-test} & control & infected & \emph{T-test} & control & infected
& \emph{T-test} & control & infected & \emph{T-test} & control &
infected & \emph{T-test}\tabularnewline
EPI & X & 8.00\selectlanguage{ngerman}±1.37 & 8.00\selectlanguage{ngerman}±2.08 & 0.99 & 8.46\selectlanguage{ngerman}±2.33 & 8.50\selectlanguage{ngerman}±1.37 & 0.92 &
9.19\selectlanguage{ngerman}±1.64 & 9.25\selectlanguage{ngerman}±2.33 & 0.91 & 10.29\selectlanguage{ngerman}±2.97 & 10.00\selectlanguage{ngerman}±1.64 & 0.65 &
12.52\selectlanguage{ngerman}±2.45 & 12.17\selectlanguage{ngerman}±4.29 & 0.58 & 14.01\selectlanguage{ngerman}± 4.29 & 14.50\selectlanguage{ngerman}±2.97 &
0.81\tabularnewline
& Y & 16.00\selectlanguage{ngerman}±4.13 & 13.00\selectlanguage{ngerman}±1.28* & 0.03 & 16.75\selectlanguage{ngerman}±12.4 & 15.00\selectlanguage{ngerman}±1.64 & 0.87 &
21.25\selectlanguage{ngerman}±3.22 & 17.10\selectlanguage{ngerman}±1.35 & 0.85 & 34.63\selectlanguage{ngerman}±11.3* & 23.93\selectlanguage{ngerman}±0.9* & 0.05 &
47.10\selectlanguage{ngerman}±8.15* & 32.10\selectlanguage{ngerman}±2.37* & 0.04 & 51.08\selectlanguage{ngerman}±9.80* & 33.82\selectlanguage{ngerman}±4.6* &
0.04\tabularnewline
& Z & 6.67\selectlanguage{ngerman}±3.01 & 5.50\selectlanguage{ngerman}±2.33 & 0.73 & 6.78\selectlanguage{ngerman}±1.69 & 5.75\selectlanguage{ngerman}±2.18 & 0.15 &
7.07\selectlanguage{ngerman}±0.33 & 6.15\selectlanguage{ngerman}±1.61 & 0.19 & 7.79\selectlanguage{ngerman}±1.39 & 6.53\selectlanguage{ngerman}±5.79 & 0.11 & 9.71\selectlanguage{ngerman}±3.54
& 6.87\selectlanguage{ngerman}±2.68 & 0.62 & 11.98\selectlanguage{ngerman}±2.13* & 7.32\selectlanguage{ngerman}±1.53* & 0.05\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & 853.8\selectlanguage{ngerman}±2.37* & 572.00\selectlanguage{ngerman}±0.3* & 0.04 &
1074.\selectlanguage{ngerman}±1.1* & 733.13\selectlanguage{ngerman}±0.3* & 0.04 & 1395.\selectlanguage{ngerman}±1.4* & 972.78\selectlanguage{ngerman}±0.4* & 0.03 &
2775\selectlanguage{ngerman}±1.7** & 1562.6\selectlanguage{ngerman}±0.5** & 0.01 & 5725.91\selectlanguage{ngerman}±1.4** & 2683.8\selectlanguage{ngerman}±0.2** & 0.01 &
8573.27\selectlanguage{ngerman}±2.26** & 3589.6\selectlanguage{ngerman}±0.4** & 0.01\tabularnewline
& Y/X & 2.00\selectlanguage{ngerman}±0.36* & 1.63\selectlanguage{ngerman}±0.71 & 0.39 & 1.98\selectlanguage{ngerman}±0.12 & 1.76\selectlanguage{ngerman}±0.81 & 0.93 &
2.30\selectlanguage{ngerman}±0.39 & 1.85\selectlanguage{ngerman}±0.11 & 0.08 & 3.33\selectlanguage{ngerman}±0.4* & 2.39\selectlanguage{ngerman}±1.25* & 0.05 &
3.77\selectlanguage{ngerman}±0.08* & 2.64\selectlanguage{ngerman}±0.35* & 0.05 & 3.64\selectlanguage{ngerman}±0.95* & 2.33\selectlanguage{ngerman}±0.28* &
0.04\tabularnewline
& percentage & 0.51\selectlanguage{ngerman}±0.11* & 0.44\selectlanguage{ngerman}±1.32* & 0.05 & 0.24\selectlanguage{ngerman}±0.30* & 0.35\selectlanguage{ngerman}±1.56*
& 0.05 & 0.25\selectlanguage{ngerman}±0.52* & 0.46\selectlanguage{ngerman}±0.49* & 0.04 & 0.33\selectlanguage{ngerman}±0.37* & 0.22\selectlanguage{ngerman}±0.61* & 0.05
& 0.39\selectlanguage{ngerman}±0.10* & 0.17\selectlanguage{ngerman}±0.46* & 0.05 & 0.27\selectlanguage{ngerman}±0.37* & 0.19\selectlanguage{ngerman}±0.47* &
0.05\tabularnewline
EN & X & 10.21\selectlanguage{ngerman}±1.78 & 14.00\selectlanguage{ngerman}±1.81 & 0.09 & 12.67\selectlanguage{ngerman}±3.69 & 14.50\selectlanguage{ngerman}±1.79 & 0.46
& 18.65\selectlanguage{ngerman}±5.36 & 15.05\selectlanguage{ngerman}±5.37 & 0.61 & 23.35\selectlanguage{ngerman}±9.75 & 19.30\selectlanguage{ngerman}±3.69 & 0.59 &
27.91\selectlanguage{ngerman}±8.42 & 23.10\selectlanguage{ngerman}±8.42 & 0.73 & 30.82\selectlanguage{ngerman}±11.51* & 24.76\selectlanguage{ngerman}±2.30 &
0.26\tabularnewline
& Y & 8.03\selectlanguage{ngerman}±1.89 & 9.50\selectlanguage{ngerman}±1.25 & 0.86 & 8.75\selectlanguage{ngerman}±1.69 & 10.00\selectlanguage{ngerman}±3.61 & 0.75 &
9.75\selectlanguage{ngerman}±1.52 & 10.75\selectlanguage{ngerman}±6.64 & 0.63 & 10.40\selectlanguage{ngerman}±2.15 & 11.75\selectlanguage{ngerman}±10.25 & 0.58 &
10.77\selectlanguage{ngerman}±1.39 & 12.33\selectlanguage{ngerman}±1.79 & 0.55 & 13.54\selectlanguage{ngerman}±4.81 & 13.42\selectlanguage{ngerman}±8.29 &
0.24\tabularnewline
& Z & 5.22\selectlanguage{ngerman}±0.26 & 5.50\selectlanguage{ngerman}±3.28 & 0.91 & 5.47\selectlanguage{ngerman}±1.21 & 5.65\selectlanguage{ngerman}±1.81 & 0.83 &
6.58\selectlanguage{ngerman}±0.27 & 5.95\selectlanguage{ngerman}±2.79 & 0.42 & 7.55\selectlanguage{ngerman}±0.51 & 6.98\selectlanguage{ngerman}±4.62 & 0.68 & 8.09\selectlanguage{ngerman}±1.05
& 8.10\selectlanguage{ngerman}±9.65 & 0.09 & 9.23\selectlanguage{ngerman}±1.67 & 6.92\selectlanguage{ngerman}±0.35 & 0.19\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & 427.9\selectlanguage{ngerman}±1.35* & 731.50\selectlanguage{ngerman}±0.6* & 0.05 &
606.4\selectlanguage{ngerman}±0.6* & 819.25\selectlanguage{ngerman}±0.2* & 0.05 & 1196.\selectlanguage{ngerman}±1.7* & 962.64\selectlanguage{ngerman}±0.6* & 0.05 &
1833.4\selectlanguage{ngerman}±1.5* & 1582.89\selectlanguage{ngerman}±0.4* & 0.04 & 2431.78\selectlanguage{ngerman}±2.33* & 2307.07\selectlanguage{ngerman}±0.4* & 0.05
& 3851.70\selectlanguage{ngerman}±3.38** & 2299.4\selectlanguage{ngerman}±0.2** & 0.01\tabularnewline
& Y/X & 0.79\selectlanguage{ngerman}±0.92* & 0.68\selectlanguage{ngerman}±0.37* & 0.05 & 0.69\selectlanguage{ngerman}±0.35 & 0.69\selectlanguage{ngerman}±0.17 & 0.66 &
0.52\selectlanguage{ngerman}±0.13* & 0.71\selectlanguage{ngerman}±0.43* & 0.05 & 0.45\selectlanguage{ngerman}±0.47 & 0.61\selectlanguage{ngerman}±0.62 & 0.06 &
0.39\selectlanguage{ngerman}±0.79* & 0.53\selectlanguage{ngerman}±0.54* & 0.05 & 0.44\selectlanguage{ngerman}±0.34* & 0.54\selectlanguage{ngerman}±0.37* &
0.05\tabularnewline
& percentage & 0.49\selectlanguage{ngerman}±0.98* & 0.56\selectlanguage{ngerman}±0.12* & 0.03 & 0.14\selectlanguage{ngerman}±0.82* & 0.39\selectlanguage{ngerman}±0.57*
& 0.04 & 0.22\selectlanguage{ngerman}±0.35* & 0.48\selectlanguage{ngerman}±0.81* & 0.03 & 0.23\selectlanguage{ngerman}±0.3* & 0.25\selectlanguage{ngerman}±0.33* & 0.05
& 0.17\selectlanguage{ngerman}±0.26 & 0.18\selectlanguage{ngerman}±0.19 & 0.29 & 0.13\selectlanguage{ngerman}±0.31* & 0.10\selectlanguage{ngerman}±0.43* &
0.05\tabularnewline
SPL & X & - & - & \textbf{-} & 13.60\selectlanguage{ngerman}±1.26 & 12.28\selectlanguage{ngerman}±3.36 & 0.26 & - & - &
\textbf{-} & - & - & \textbf{-} & - & - & \textbf{-} & - & - &
\textbf{-}\tabularnewline
& Y & - & - & \textbf{-} & 12.70\selectlanguage{ngerman}±1.11 & 10.79\selectlanguage{ngerman}±2.18 & 0.39 & - & - &
\textbf{-} & - & - & \textbf{-} & - & - & \textbf{-} & - & - &
\textbf{-}\tabularnewline
& Z & - & - & \textbf{-} & 8.98\selectlanguage{ngerman}±1.25 & 8.18\selectlanguage{ngerman}±5.19 & 0.51 & - & - &
\textbf{-} & - & - & \textbf{-} & - & - & \textbf{-} & - & - &
\textbf{-}\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & - & \textbf{-} & 1551.\selectlanguage{ngerman}±3.4* &
1083.9\selectlanguage{ngerman}±0.2* & 0.03 & - & - & \textbf{-} & - & - & \textbf{-} & - & - &
\textbf{-} & - & - & \textbf{-}\tabularnewline
& Y/X & - & - & \textbf{-} & 0.28\selectlanguage{ngerman}±1.3* & 0.88\selectlanguage{ngerman}±0.11* & 0.05 & - & - &
\textbf{-} & - & - & \textbf{-} & - & - & \textbf{-} & - & - &
\textbf{-}\tabularnewline
& percentage & - & - & \textbf{-} & 0.27\selectlanguage{ngerman}±0.98* & 0.16\selectlanguage{ngerman}±0.52* & 0.04 & - &
- & \textbf{-} & - & - & \textbf{-} & - & - & \textbf{-} & - & - &
\textbf{-}\tabularnewline
ML & X & - & - & \textbf{-} & - & - & \textbf{-} & 6.72\selectlanguage{ngerman}±1.83 & 6.45\selectlanguage{ngerman}±0.88
& 0.26 & 7.30\selectlanguage{ngerman}±2.91 & 8.18\selectlanguage{ngerman}±1.52 & 0.28 & - & - & \textbf{-} & - & - &
\textbf{-}\tabularnewline
& Y & - & - & \textbf{-} & - & - & \textbf{-} & 6.75\selectlanguage{ngerman}±1.72 & 5.85\selectlanguage{ngerman}±2.36 &
0.17 & 7.08\selectlanguage{ngerman}±2.38 & 6.30\selectlanguage{ngerman}±3.17 & 0.18 & - & - & \textbf{-} & - & - &
\textbf{-}\tabularnewline
& Z & - & - & \textbf{-} & - & - & \textbf{-} & 5.00\selectlanguage{ngerman}±0.35 & 4.5\selectlanguage{ngerman}±2.59 &
0.31 & 5.22\selectlanguage{ngerman}±0.24 & 6.68\selectlanguage{ngerman}±0.99 & 0.82 & - & - & \textbf{-} & - & - &
\textbf{-}\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & - & \textbf{-} & - & - &
\textbf{-} & 226.8\selectlanguage{ngerman}±2.3* & 169.79\selectlanguage{ngerman}±0.4* & 0.03 & 269.79\selectlanguage{ngerman}±1.4* & 344.25\selectlanguage{ngerman}±0.3*
& 0.05 & - & - & \textbf{-} & - & - & \textbf{-}\tabularnewline
& Y/X & - & - & \textbf{-} & - & - & \textbf{-} & 0.79\selectlanguage{ngerman}±0.33* &
0.91\selectlanguage{ngerman}±0.63* & 0.05 & 0.81\selectlanguage{ngerman}±0.88 & 0.77\selectlanguage{ngerman}±0.31 & 0.22 & - & - & \textbf{-} &
- & - & \textbf{-}\tabularnewline
& percentage & - & - & \textbf{-} & - & - & \textbf{-} & 0.04\selectlanguage{ngerman}±0.26 &
0.49\selectlanguage{ngerman}±0.44 & 0.24 & 0.04\selectlanguage{ngerman}±0.35 & 0.36\selectlanguage{ngerman}±0.26 & 0.18 & - & - & \textbf{-} & -
& - & \textbf{-}\tabularnewline
TA & X & - & - & \textbf{-} & - & - & \textbf{-} & 7.39\selectlanguage{ngerman}±2.65 & 7.40\selectlanguage{ngerman}±1.47
& 0.16 & 7.94\selectlanguage{ngerman}±3.59 & 8.55\selectlanguage{ngerman}±2.91 & 0.38 & 8.43\selectlanguage{ngerman}±8.22 & 8.00\selectlanguage{ngerman}±3.03 & 0.84 & -
& - & \textbf{-}\tabularnewline
& Y & - & - & \textbf{-} & - & - & \textbf{-} & 7.10\selectlanguage{ngerman}±3.34 & 5.95\selectlanguage{ngerman}±2.35 &
0.17 & 7.80\selectlanguage{ngerman}±4.07 & 6.85\selectlanguage{ngerman}±0.88 & 0.38 & 8.35\selectlanguage{ngerman}±6.73 & 7.60\selectlanguage{ngerman}±1.52 & 0.23 & - &
- & \textbf{-}\tabularnewline
& Z & - & - & \textbf{-} & - & - & \textbf{-} & 6.31\selectlanguage{ngerman}±1.85 & 6.25\selectlanguage{ngerman}±3.26 &
0.24 & 8.64\selectlanguage{ngerman}±1.58 & 6.38\selectlanguage{ngerman}±9.61 & 0.41 & 6.32\selectlanguage{ngerman}±1.43 & 6.80\selectlanguage{ngerman}±3.33 & 0.75 & - &
- & \textbf{-}\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & - & \textbf{-} & - & - &
\textbf{-} & 331.0\selectlanguage{ngerman}±3.0* & 275.19\selectlanguage{ngerman}±0.8* & 0.05 & 535.09\selectlanguage{ngerman}±1.4* & 373.66\selectlanguage{ngerman}±0.2*
& 0.04 & 444.87\selectlanguage{ngerman}±2.07* & 413.44\selectlanguage{ngerman}±0.26 & 0.09 & - & - &
\textbf{-}\tabularnewline
& Y/X & - & - & \textbf{-} & - & - & \textbf{-} & 0.96\selectlanguage{ngerman}±0.07 & 0.80\selectlanguage{ngerman}±0.35
& 0.06 & 0.98\selectlanguage{ngerman}±0.28* & 0.80\selectlanguage{ngerman}±0.31* & 0.05 & 0.99\selectlanguage{ngerman}±0.72 & 0.95\selectlanguage{ngerman}±0.50 & 0.91 &
- & - & \textbf{-}\tabularnewline
& percentage & - & - & \textbf{-} & - & - & \textbf{-} & 0.06\selectlanguage{ngerman}±0.66* &
0.75\selectlanguage{ngerman}±0.51* & 0.03 & 0.07\selectlanguage{ngerman}±0.34* & 0.41\selectlanguage{ngerman}±0.49* & 0.05 & 0.04\selectlanguage{ngerman}±0.58** &
0.23\selectlanguage{ngerman}±0.37** & 0.01 & - & - & \textbf{-}\tabularnewline
PMC & X & - & - & \textbf{-} & 11.86\selectlanguage{ngerman}±2.3* & 19.98\selectlanguage{ngerman}±3.59* & 0.05 &
12.6\selectlanguage{ngerman}±3.03* & 23.70\selectlanguage{ngerman}±4.4* & 0.05 & - & - & - & - & - & - & - & - &
-\tabularnewline
& Y & - & - & \textbf{-} & 11.58\selectlanguage{ngerman}±1.44 & 16.85\selectlanguage{ngerman}±1.27 & 0.18 & 12.29\selectlanguage{ngerman}±2.9* &
20.35\selectlanguage{ngerman}±2.6* & 0.05 & - & - & - & - & - & - & - & - & -\tabularnewline
& Z & - & - & \textbf{-} & 9.73\selectlanguage{ngerman}±1.36 & 7.45\selectlanguage{ngerman}±5.37 & 0.71 & 10.73\selectlanguage{ngerman}±2.09 &
8.95\selectlanguage{ngerman}±1.39 & 0.61 & - & - & - & - & - & - & - & - & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & - & \textbf{-} & 1336\selectlanguage{ngerman}±5** &
2508.\selectlanguage{ngerman}±0.6** & 0.01 & 1658\selectlanguage{ngerman}±1.5** & 4316\selectlanguage{ngerman}±0.6** & 0.01 & - & - & - & - & -
& - & - & - & -\tabularnewline
& Y/X & - & - & \textbf{-} & 0.98\selectlanguage{ngerman}±0.09 & 0.84\selectlanguage{ngerman}±0.56 & 0.77 & 0.98\selectlanguage{ngerman}±0.46 &
0.86\selectlanguage{ngerman}±0.25 & 0.06 & - & - & - & - & - & - & - & - & -\tabularnewline
& percentage & - & - & \textbf{-} & 0.31\selectlanguage{ngerman}±0.18* & 44.33\selectlanguage{ngerman}±0.17* & 0.03 &
0.29\selectlanguage{ngerman}±0.35* & 0.56\selectlanguage{ngerman}±0.48* & 0.02 & - & - & - & - & - & - & - & - &
-\tabularnewline
MP & X & - & - & - & - & - & - & - & - & - & 15.33\selectlanguage{ngerman}±5.78* & 26.40\selectlanguage{ngerman}±1.3* &
0.05 & 19.60\selectlanguage{ngerman}±4.31 & 28.80\selectlanguage{ngerman}±2.58* & 0.04 & - & - & -\tabularnewline
& Y & - & - & - & - & - & - & - & - & - & 14.76\selectlanguage{ngerman}±5.65* & 23.33\selectlanguage{ngerman}±8.7* &
0.04 & 20.05\selectlanguage{ngerman}±1.64 & 25.47\selectlanguage{ngerman}±2.55 & 0.59 & - & - & -\tabularnewline
& Z & - & - & - & - & - & - & - & - & - & 12.13\selectlanguage{ngerman}±5.51 & 11.48\selectlanguage{ngerman}±4.62 & 0.53
& 15.33\selectlanguage{ngerman}±5.97 & 13.67\selectlanguage{ngerman}±0.99 & 0.07 & - & - & -\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & - & - & - & - & - & - & - & - &
2744\selectlanguage{ngerman}±3.07** & 7070.6\selectlanguage{ngerman}±0.4** & 0.01 & 6024.38\selectlanguage{ngerman}±5.31 & 10027.4\selectlanguage{ngerman}±0.3* & 0.03 &
- & - & -\tabularnewline
& Y/X & - & - & - & - & - & - & - & - & - & 0.96\selectlanguage{ngerman}±0.81* & 0.88\selectlanguage{ngerman}±0.39* &
0.05 & 1.02\selectlanguage{ngerman}±0.42* & 0.88\selectlanguage{ngerman}±0.49* & 0.05 & - & - & -\tabularnewline
& percentage & - & - & - & - & - & - & - & - & - & 0.33\selectlanguage{ngerman}±0.48** &
0.59\selectlanguage{ngerman}±0.72** & 0.01 & 0.41\selectlanguage{ngerman}±0.35** & 0.66\selectlanguage{ngerman}±0.21** & 0.01 & - & - &
-\tabularnewline
PG & X & - & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
28.91\selectlanguage{ngerman}±5.34 & 31.92\selectlanguage{ngerman}±0.18 & 0.19\tabularnewline
& Y & - & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
30.46\selectlanguage{ngerman}±5.18* & 27.94\selectlanguage{ngerman}±4.3* & 0.05\tabularnewline
& Z & - & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
25.15\selectlanguage{ngerman}±6.19 & 15.88\selectlanguage{ngerman}±2.83 & 0.17\tabularnewline
& Volume (\selectlanguage{greek}μ\selectlanguage{english}m\textsuperscript{3}) & - & - & - & - & - & - & - & - & - & -
& - & - & - & - & - & 22147.1\selectlanguage{ngerman}±0.97** & 14162\selectlanguage{ngerman}±0.2** & 0.01\tabularnewline
& Y/X & - & - & - & - & - & - & - & - & - & - & - & - & - & - & - &
1.05\selectlanguage{ngerman}±0.31* & 0.88\selectlanguage{ngerman}±0.57* & 0.05\tabularnewline
& percentage & - & - & - & - & - & - & - & - & - & - & - & - & - & - & -
& 0.60\selectlanguage{ngerman}±0.38* & 0.72\selectlanguage{ngerman}±0.44* & 0.02\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3-/Figure-3-}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-4-/Figure-4-}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-5-/Figure-5-}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-6-/Figure-6-}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-7-/Figure-7-}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-8/Figure-8}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-9-/Figure-9-}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-10/Figure-10}

\end{center}
\end{figure}

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\selectlanguage{english}
\FloatBarrier
\end{document}

