Histopathological assessment of the infection of maize leaves by Fusarium graminearum, F. proliferatum, and F. verticillioides

Histopathological assessment of the infection of maize leaves by Fusarium graminearum, F. proliferatum, and F. verticillioides

Accepted Manuscript Histopathological assessment of the infection of maize leaves by Fusarium graminearum, F. proliferatum and F. verticillioides Thi ...

6MB Sizes 1 Downloads 18 Views

Accepted Manuscript Histopathological assessment of the infection of maize leaves by Fusarium graminearum, F. proliferatum and F. verticillioides Thi Thanh Xuan Nguyen, Heinz-Wilhelm Dehne, Ulrike Steiner PII:

S1878-6146(16)30064-2

DOI:

10.1016/j.funbio.2016.05.013

Reference:

FUNBIO 727

To appear in:

Fungal Biology

Received Date: 14 July 2015 Revised Date:

9 April 2016

Accepted Date: 31 May 2016

Please cite this article as: Nguyen, T.T.X., Dehne, H.-W., Steiner, U., Histopathological assessment of the infection of maize leaves by Fusarium graminearum, F. proliferatum and F. verticillioides, Fungal Biology (2016), doi: 10.1016/j.funbio.2016.05.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Histopathological assessment of the infection of maize leaves by Fusarium graminearum, F. proliferatum and F. verticillioides

RI PT

Thi Thanh Xuan NGUYENa,b, Heinz-Wilhelm DEHNEa, Ulrike STEINERa a

Institute of Crop Science and Resource Conservation, Division Phytomedicine, University of Bonn, Nussallee 9, 53115 Bonn, Germany

b

Corresponding author: Thi Thanh Xuan NGUYEN Tel: +84 1275535643

M AN U

Email: [email protected]

SC

Faculty of Agriculture and Natural Resources, University of An Giang, 18 Ung Van Khiem, An Giang, Vietnam

Abstract

AC C

EP

TE D

Young maize plants were inoculated on unfolded mature leaves and on folded immature leaves with Fusarium graminearum, F. proliferatum and F. verticillioides suspensions. Infection and symptom development of disease on these asymptomatic mature leaves and immature leaves were then documented. Subcuticular infection was found by the three Fusarium species on both symptomatic and symptomless leaves. The three Fusarium species penetrated the stomata of immature leaves by the formation of appressoria-like structures, infection cushions or by direct penetration. Infection by the three species of Fusarium via stomata is reported here for the first time. The superficial hyphae and re-emerging hyphae of the three species produced conidia. The macroconidia of F. graminearum produced secondary macroconidia and F. proliferatum formed microconidia inside the leaf tissues that sporulated through stomata and trichomes. The infection of maize leaves by the three species of Fusarium and their sporulation may contribute inoculum to cob and kernel infection. Key words: Fusarium spp., stomata, sporulation, symptom, penetration, foliage infection

1. Introduction

Several fungal species belonging to the genus Fusarium are known to constrain cereal production in many regions of the world. Among the economically important diseases of cereal crops caused by Fusarium spp. are root, stem and ear rot of maize, Fusarium head blight (FHB) 1

ACCEPTED MANUSCRIPT

RI PT

and crown rot (Koehler 1942; Burgess et al. 1981; Parry et al. 1995; Doohan et al. 2003; Williams et al. 2007; Görtz et al. 2010). The infection of cereals by such fungi causes significant yield losses, both in quantity and grain quality (Klein et al. 1991; Southwell et al. 2003). In general, many fungi including species of Fusarium are mycotoxin producers. Apart from causing a variety of health problems in humans and animals, some of the mycotoxins have been reported to play a role in pathogen virulence during infection of the plant (Lamprecht et al. 1994; Desjardins et al. 1998). Moreover, yield losses and reduction in grain quality have been observed to be related to the amount of mycotoxin produced in a particular grain by such fungi (McMullen et al. 1997).

M AN U

SC

Many studies have been conducted worldwide to distinguish between the different types of diseases caused by Fusarium. For instance, Logrieco et al. (2002) concluded that maize ear rots caused by Fusarium spp. can be categorized as pink and red ear rot. The authors further showed that F. verticillioides, F. proliferatum and F. subglutinans were the causative agents of pink ear rot, while F. graminearum, F. culmorum, F. cerealis and F. avenaceum were often associated with red ear rot (Logrieco et al. 2002). However, the occurrence and appearance of these diseases often depend on environmental conditions. Pink ear rot, for instance, occurred frequently in temperate regions with cooler climates (Munkvold and Desjardins 1997) while red ear rot was often found in regions that experience high humidity or rainfall and moderate temperatures (Logrieco et al. 2002; Munkvold 2003). However, species of Fusarium have also been considered symptomless endophytes of maize (Thomas 1980; Bacon and Hinton 1996; Munkvold et al. 1997a; Bacon et al. 2008).

AC C

EP

TE D

Like many other fungi, Fusarium can infect hosts in various ways. In maize, Fusarium infection can take place systemically or locally (Sutton 1982; Parry et al. 1995; Munkvold and Desjardins 1997). In systemic infection, fungal hyphae usually grow from infected seeds colonize the stalk and then the kernel (Lawrence 1981). Local infection through silks has been reported to play an important role in kernel infection (Reid 1992; Chungu 1996; Munkvold et al. 1997b; Reid 2002). In most cases, the fungal hyphae play a significant role in host infection due to their ability to produce enzymes that degrade host cell walls (Kang and Buchenauer 2000b). In addition, Lawrence (1981) demonstrated that F. verticillioides hyphae entered through the xylem of leaf and stem tissues. Infection of floral organs of wheat by F. graminearum has also been reported, and infection began with the formation of foot structures, lobate appressoria and infection cushions (Boenisch and Schäfer 2011). On the other hand, F. culmorum hyphae were noted to penetrate different parts of wheat spikelets and sometimes via stomata (Kang and Buchenauer 2000a). The causative agents of FHB and ear rot produce many airborne conidia that aid in dispersal and host invasion. Conidia can be windblown or rain splashed on to the silks or spikelets prior to the infection of the kernels (Sutton 1982, Munkvold and Desjardins 1997; Trail 2009). Only a few studies have examined the infection and colonization of maize plants by species of Fusarium through the leaves (Wagacha et al. 2012). However, no information exists on the process associated with the spread of Fusarium conidia from the shoot to the silks and then to the cobs of maize following leaf colonization. This information would be important in assessing plant health and safety during the early stages of growth and add to our knowledge of the epidemiology of the disease. This study, therefore, aimed at investigating the histopathological 2

ACCEPTED MANUSCRIPT

processes involved in the infection of maize leaves by Fusarium graminearum, F. proliferatum and F. verticillioides.

Fungal pathogen and inoculum preparation

RI PT

2. Materials and methods Fusarium proliferatum (Matsushima) Nirenberg, isolate AG31g, F. verticillioides (Sacc.) Nirenberg, isolate AG11i, and F. graminearum isolate AG 23d were used in the study. The isolates were originally obtained from maize kernels collected in Germany (Görtz et al. 2008).

TE D

M AN U

SC

Pure cultures were grown and maintained on Potato Dextrose Agar (39 g/L PDA, Sigma Aldrich Chemie GmbH, Taufkirchen, Germany) in Petri dishes and then incubated at 22 °C for at least 7 days. Then two fungal plugs (Ø 1 cm) were cut from the 7-day old cultures and added to Potato Dextrose Broth (39 g/L PDB, Sigma - Aldrich Chemie GmbH, Taufkirchen, Germany) media in 500 mL Erlenmeyer flasks containing 100 mL of media. The cultures were incubated on a shaker at 120 rpm, 22 °C and in total darkness for 3-4 days. A 0.5 mL fungal suspension was then spread on the surface of low-strength Potato Dextrose Agar (12.5 g/ L PDA, 19 g/L Agar- agar) media. Inoculated Petri dishes were air-dried under a laminar flow cabinet for 10-20 minutes (mins) then incubated under conditions of near ultra violet light at 22 °C for 3-5 days (Moradi 2008). Conidia were harvested by flooding the plates with sterile distilled water containing Tween 20 (0.075 %) followed by slight scraping with a spatula. The suspension was sieved through double-layered cheesecloth. The concentration of the conidia was determined using a Fuchs-Rosenthal chamber and then adjusted to 2x106 spores/mL.

Maize cultivation, inoculation and sampling

AC C

EP

Seeds of the maize cv. Tassilo were disinfected with hot water (Rahman et al. 2008). The seeds were soaked in water for 4 hours (hrs) at room temperature and then treated in hot water at 50-52 °C for 15 mins. The seeds were then sown in trays in Klasmann potting substrate (Klasmann-Deilmann, Geeste, Germany). After germination, uniform seedlings were selected and transplanted, one plant per pot in the same substrate (Klasmann potting). The plants were placed in a growth chamber and the surface of the pots carefully watered once a day to avoiding water sprinkling onto the foliage. Fifteen day old maize plants were inoculated on the 4th mature unfolded leaf and on the 6th folded immature leaf. Thirty maize plants were inoculated separately with each Fusarium species then incubated in growth chambers under high humidity (90-95 %) for 48 hrs and then maintained in a growth chamber at a temperature ranging from 18-20 °C and 22-24 °C, and a relative humidity from 45-55 % and 75-83 %, respectively during the day and night. A photoperiod of 15 hrs was applied. For each sampling date, eight 1 cm2 leaf segments (four from the 4th leaf and four from the 6th leaf) per treatment were collected every day until the 30th day after inoculation (dai). 3

ACCEPTED MANUSCRIPT

Measurement of infected stomata

RI PT

The number of infected stomata was counted on four 1 cm2 leaf segments (the 6th leaf) per treatment from 7 and 15 dai by using a Leitz DMR photomicroscope (Leica, Wetzlar, Germany).

Light microscopy Leaf sections

SC

Leaf sections were cut out of the leaves and mounted on a microscope slide in diethanol (0.01 %) and covered with a cover slip. The specimens were observed with the BP 340380/FT 400/LP 430 filter combination using a Leitz microscope in the fluorescence mode. Images were photographed with a fitted digital camera and saved using the program “Discus” (Technisches Büro Hilgers, Koenigswinter, Germany).

M AN U

Whole specimen Clearing specimens:

Staining:

TE D

Sampled leaf sections were cut from the leaves and soaked immediately in saturated chloral hydrate solution (250 g/100 mL H2O). To reduce washing off of conidia from the leaf surface, 0.01 % formaldehyde was added to the chloral hydrate solution before use. For full clearance of the chlorophyll, leaf pieces were left in the clearing solution for 7 days at room temperature. After the sections were cleared of chlorophyll, they were stained and examined with a light microscope.

AC C

Observation:

EP

For staining of whole specimens, the cleared leaf samples were immediately immersed in a solution according to Bruzzese and Hasan (1983) (300 mL 95 % ethanol, 150 mL chloroform, 125 mL 90 % lactic acid, 450 g chloral hydrate and 0.6 g aniline blue) for 24 hrs or they were placed in fuchsin acid (10 mL phenol, 10 mL glycerine, 10 mL lactic acid and 3 mg fuchsin acid) for 48 hrs. Fungal structures were stained blue when stained with Bruzzese and Hasan solution and pink with fuchsin solution.

Stained samples were mounted on microscope slides and then covered with a cover slip and observed with Nomarski-interference-contrast using Leitz DMR photomicroscope. Images were photographed with a digital camera and saved using the program “Discus” (Technisches Büro Hilgers, Koenigswinter, Germany). Scanning electron microscopy Leaf pieces ~ 1 cm2 size were directly mounted on SEM stubs and coated with gold by an automatic sputter coater MSC1 S/N 201106 and then observed with a Phenom World SEM (Fei, Eindhoven, Netherlands). Transmission electron microscopy 4

ACCEPTED MANUSCRIPT

Fixing specimens:

RI PT

Leaf tissues showing lesion symptoms were cut into pieces of 2 x 2 mm2 and fixed by mixing 2 % paraformaldehyde, 2 % glutaraldehyde and 0.03 % calcium chloride in 0.2 M cacodylic acid sodium salt trihydrate buffer, pH 7.3-7.5 (Karnovsky, 1965) for 4 hrs at room temperature. The samples were then washed by placing them in cacodylic acid sodium salt trihydrate buffer for 10 mins. The washing was repeated nine times and thereafter the tissues were post-fixed in 1% osmium tetroxide (OsO4) for 2 hrs. The tissues were then rinsed eight times in cacodylic acid sodium salt trihydrate buffer (pH 7.35), for 15 mins each rinse. Samples were dehydrated in increasing concentrations of ethanol 15, 30, 50, 70, 80, 90 and 100 % for 15 mins in each concentration.

M AN U

SC

The dehydrated samples were next washed in 2 changes of propylene oxide, each wash lasting for 10 mins and then infiltrated with different ratios of agar low viscosity resin (Agar Scientific Ltd.) and propylene oxide. The agar low viscosity resin comprised of 48 g LV resin, 16 g VH1 hardener, 36 g VH2 hardener and 2.5 g LV accelerator. The concentrations of agar low viscosity resin in relation to propylene oxide were: 1:3, 1:1, 3:1 and 1:0. For each concentration, the infiltration process lasted 22 hrs. The samples were subsequently polymerized in 100 % agar low viscosity resin in flat embedding trays (Agar-Aids) at 60 °C for 24 hrs. Sectioning and observing:

TE D

Semi-thin sections: Polymerized sample blocks were cut by using a 45° glass knife, directly suspended in distilled water then transferred onto a glass slide and dried on an electric plate at 70 °C. Dried leaf sections on the glass slides were stained in 0.5 % toluidine blue (w/v) in 0.01 M phosphate buffer (pH 7.4). The sections were washed in tap water and in distilled water to removed excess stain and dried on an electric plate at 70 °C. Stained sections were placed in xylene for 5 mins, mounted and sealed in an entellan rapid mounting media (Merck, Darmstadt, Germany) and then air-dried overnight in a fume chamber before being viewed under light microscope.

AC C

EP

Ultra-thin sections and contrasting: If the desired fungal structures were found in the semi-thin sections, ultra-thin sections were continuously cut out of the same block. The ultra-thin sections were cut with a Reichert-Jung Ultramicrotome Ultracut E to a thickness of 70-72 nm using a diamond knife. Ultra-thin sections were placed on copper or nickel grids followed by the contrasting process (Geyer, 1973). The grids were laid out in drops of saturated 2% uranyl acetate for 8 mins, rinsed twice in bi-distilled water and then placed in drops of lead acetate solution (1.33 g Pb(NO3)2, 1.76 g Na3(C6H5O7).2H2O and 30 mL bi-distilled water) for 2 mins and rinsed in 2 changes of bi-distilled water, air dried at room condition before being stored in a grid box. The ultra-thin sections were observed with a Zeiss EM 109 transmission electron microscope (Carl Zeiss, Wetzlar, Germany) and images were photographed with a K- Frametransfer CCD camera for EM 109 and saved using the program images Sys Prog (Tröndle Restlichtverstarstärkersysteme, Germany). Data analysis

5

ACCEPTED MANUSCRIPT

3. Results

RI PT

All data were tested for normality and homogeneity of variance using Kolmogorov or ShapiroWilk tests prior to subjecting them to analysis of variance (ANOVA). Where significant differences occurred across treatments, comparisons of means were then performed by using Duncan's test at the 5% level of significance; a n IRRISTAT statistical package (version 5.0, International Rice Research Institute) was used to analyze the data. No symptoms appeared when inoculates were applied to the unfolded 4th mature leaf, whereas disease symptoms occurred on the folded 6th immature leaf at the time of its emergence. 3.1. Asymptomatic infection of the 4th unfolded mature leaf

M AN U

SC

The hyphal tips became enlarged and rounded off (appressoria - like structures) 3 dai with F. graminearum (Fig. 1a) and the hyphae grew along epidermal cells or grew in a fascicle of parallel hyphae (Fig. 1b, c). Subcuticular infection of epidermal cells was detected for F. proliferatum and subcuticular hyphae were clearly observed to grow in a coral shape starting at the corner of a cell (Fig. 1d, e). F. verticillioides hyphae colonized inter-cellular mesophyll cells However, penetration points were not clearly identifiable (Fig. 1f). Invasion of epidermal cells and inter-cellular mesophyll cells by three Fusarium species was observed at 7 dai. 3.2. Infection of immature leaves with symptoms Subcuticular, inter and intracellular infection

EP

TE D

F. graminearum hyphae invaded the cuticle through the corner of cell walls and then spread along the cell walls of the bulliform cells, thin – walled and large cells in the leaf epidermis (Fig. 2a). The subcuticle of short epidermal cells was also infected and F. graminearum infection spread into adjacent cells (Fig. 2b). On leaves showing strong symptom development, the three Fusarium species were observed to grow through intercellular spaces (Fig. 2c, d, f and g). On leaves showing mild disease symptoms, F. verticillioides hyphae colonizing intercellular spaces were clearly observed with a definite cell wall thickening (Fig. 2k). These symptoms were observed 7 dai. Intracellular infection of parenchyma cells was only observed for all three species of Fusarium in heavily infected tissues (Fig. 2e and h). Heavily infected inter- and intracellular cells (parenchyma and epidermal cells) led to distortion or collapse of the cells and adjacent cells (Fig. 2c - e, g and h).

AC C

Infection through stomata and colonization of maize leaves Different modes of infection were employed for successful penetration into maize leaves via the stomata by F. graminearum. The modes were: (1) formation of appressorium-like structures (swelling at a hyphal tip) that originated from a single hypha upon coming into contact with the stomatal aperture (Fig. 3a) and the observation of an imprint circle underneath the appressorium (Fig. 3c); and (2) formation of a hyphal cushion on the stomata surface (Fig. 3b). The process of hyphae adhering to and penetrating stomata was observed 3 dai on the immature maize leaves. After penetration through the stomatal aperture, the hyphae colonized the substomatal cavity, i.e. the biggest airspace in maize leaf tissue (Fig. 3 d). Infection of F. proliferatum via the stomata was observed beginning the 4th dai. Fungal hyphae penetrated either directly through the stomata (Fig. 3e) or formed a hyphal cushion on the 6

ACCEPTED MANUSCRIPT

RI PT

stomatal aperture (Fig. 3f, 4b). Later the hypha elongated and penetrated through the stomatal aperture into the substomatal cavity (Fig. 4c). After reaching the cavity, septate hyphae were noted to enlarge, branch and colonize the substomatal cavity (Fig. 4b, c, d, and e). Following colonization of the cavity, the hyphae invaded intercellular spaces between the parenchyma cells (Fig. 4b). From the 9 dai, fungal hyphae were observed to re-emerge from necrotic stomata or from necrotic tissue (Fig. 3h). Re-emergence of hyphae at the non- necrotic tissue was also observed at 25 dai.

Comparison of infected stomata by Fusarium species

SC

Fusarium verticillioides infected the stomata using either single or multiple germ tube(s) (Fig. 3i, j, k). Both single and multiple germ tubes swelled at the tip and came into contact with the stomatal surface. After penetration, hyphae colonized the substomatal cavity, and then grew downwards into the spaces among parenchyma cells (Fig. 3l).

M AN U

Although all three fungal species had the ability to infect the stomata of the maize leaves, the penetration levels were significantly different among the three species (Fig. 5). Infection of the stomata by F. proliferatum was highest and significantly different from F. graminearum and F. verticillioides 7 dai (P = 0.004) and 15 dai (P = 0.001, Fig. 5). 3.3. Sporulation

TE D

Sporulation occurred from both superficial hyphae, i.e. mycelia growing from applied inoculum, and from re-emerging hyphae, i.e. secondary infection hyphae. Whereas sporulation from superficial hyphae of F. graminearum was detected 48 hai, spores produced from re-emerging hyphae were observed 15 dai. Conidiophores were either single or fascicled and bore single macroconidia (Fig. 6a, b). Macroconidia were either lacking or only occasionally observed from very short single conidiophore (Fig. 6c, d).

AC C

EP

Growth and sporulation of F. proliferatum were very proliferous, with both the microconidia and macroconidia observed on the leaf surfaces as well as inside infected tissues. Microconidia produced from secondary infection hyphae arising from infected trichomes (Fig. 6j) and from necrotic lesions (Fig. 6i) were detected 9 dai. Three to six celled macroconidia were observed on the leaf surface at 15 dai (Fig. 6e). Internal sporulation was noted at 7 dai. Microconidia arose from hyphae colonizing dead necrotic internal tissues and from the stomatal cavity (Fig. 6f, g). Microconidia were released from the stomatal aperture and trichomes (Fig. 6h). Similarly, F. verticillioides produced conidia either from superficial hyphae or from secondary infection hyphae. Microconidia formed from superficial hyphae (Fig. 6l) and from germ tubes (Fig. 6k). However, sporulation of F. verticillioides from superficial hyphae was detected much later than for F. graminearum and F. proliferatum, i.e. 3 dai. 4. Discussions

Many Fusarium species are known to have an endophytic phase during colonization of the host (Bacon and Hinton 1996; Yates et al. 1997; Yates et al. 1999; Vieira 2000; Oiah et al. 2006; Larran et al. 2007) by which the fungi penetrated and colonized host cells without damaging host tissue. In the present investigation, hyphae of all three Fusarium species were found in symptomless leaves. Direct penetration and appressorium formation were observed with F. 7

ACCEPTED MANUSCRIPT

RI PT

graminearum, whereas F. proliferatum and F. verticillioides penetrated directly. These findings are in line with previous research findings in which F. moniliforme penetrated maize roots directly (Lawrence 1981) and infected symptomless maize plants (Bacon and Hinton 1996). They reported that hyphae colonized intercellular spaces of secondary and primary roots and internodes from systemic infection. The results of the present study revealed that F. graminearum, F. proliferatum and F. verticillioides infected maize leaves locally and further invaded the leaf tissue either inter - or intra - cellularly. F. graminearum invaded epidermal cells whereas F. proliferatum and F. verticillioides occupied mesophyll cells. However, at an infection site only one to three cells were colonized, adjacent cells were not infected by the three fungi. These results are similar to those of Schulz (1999) who stated that Fusarium sp. colonized barley (Hordeum vulgare L.) inter- and intracellularly.

TE D

M AN U

SC

The penetration sites of asymptomatic infections by Fusarium were not conspicuous (Wagacha et al. 2012). In this study, hyphae grew randomly or along grooved /anticlinal cell walls on the surface of the host leaf tissue, formed mycelia networks and then penetrated the maize leaf. The penetration point for F. proliferatum and F. verticillioides were not clearly observed, but the penetration site of F. graminearum was usually dectected at the corner of cell walls following the formation of an appressorium-like structure. These characteristics could be explained by morphological and biological properties of the leaf. For example, hyphae may adhere to the grooves/anticlines easier than smooth surfaces; or hyphae may have been capable of perceiving signals of water, free spaces and nutrients from those sites. Intercellular endophytic colonization requires nutrients from the apoplast for growth (Schulz and Boyle 2005). Nutrients within apoplast host cells were diverse and plentiful for fungi growth and reproduction (Canny 1995; Bacon and White 2000; Tejera et al. 2006). Knight (2011) also found hyphae of F. pseudograminearum growing along grooves of the wheat sheath surface.

AC C

EP

However, species of Fusarium are also known to form symptoms after infection (Ding et al. 2011). Fusarium spp. caused symptoms on the kernels, crowns, leaf sheaths, and stems of maize plants (Gilbertson et al. 1985; Gilbertson 1986; Hampton et al. 1997; Munkvold and Desjardins 1997; Pastirčák 2004; Reid 2002; Santiago et al. 2007; Dutton 2009;). The results obtained in the present study showed that symptoms only appeared when the immature folded leaves were inoculated. On infected leaves with disease symptoms, there were two typical infection patterns of the species tested: F. graminearum, F. proliferatum and F. verticillioides: (1) infection into trichomes of maize leaves by Fusarium was described (unpublished data) and (2) infection of stomata was a strategy the fungi used to infect the host in the present studies. Knight (2011) also reported that F. pseudograminearum frequently infected stomata of wheat leaf sheath tissue that resulted in lesion formation. In the present study, the three species of Fusarium infected maize leaves via stomata. This infection pathway occurred frequently on inoculated immature leaves but was not observed on inoculated mature leaves. This may be explained by the fact that the stomata of immature leaves may be open more often because of the high humidity conditions found within the whorl. Although the three Fusarium species in this study had the capacity to infect via stomata, they showed different patterns of behaviour. F. graminearum formed appressorium-like structures or infection cushions, or even penetrated directly. The formation of an infection cushion or direct penetration was also seen with F. proliferatum. Direct penetration or the formation of appressorium-like structures was observed 8

ACCEPTED MANUSCRIPT

RI PT

with F. verticillioides. These findings are in line with research of Boenisch and Schäfer (2011) in which F. graminearum formed lobate appressoria and infection cushions to penetrate caryopses, paleas, lemmas, and glumes of wheat plants. On the other hand, F. moniliforme was observed to directly penetrate epidermal cells of seedling maize (Lawrence 1981; Murillo et al. 1999). Kang and Buchenauer (2000a) found that F. culmorum occasionally penetrated via stomata on the inner surface of the lemma of wheat spikes.

M AN U

SC

Another mechanism by which the three species of Fusarium gained entry into the leaf tissues was through the cuticle. Upon penetration the fungus was observed to grow and colonize the subcuticular spaces of short epidermal cells. In other investigations F. graminearum and F. culmorum hyphae were observed to grow inside the subcuticles of glume, palea, and lemma of wheat spikes (Kang and Buchenauer 2000a; Pritsch et al. 2000; Jansen et al. 2005). These species may penetrate the cuticle with short infection hypha (Mary Wanjiru et al. 2002) or secrete enzyme that degrade cuticle (Kang and Buchenauer 2000b). In the present study hyphae of Fusarium were observed under the cuticle of epidermal cells, particularly in short epidermal cells.

AC C

EP

TE D

Fungal sporulation facilitates the dispersal and preservation of fungal species. Pritsch et al. (2000) observed F. graminearum sporulation within 48 to 76 hai on inoculated glumes. Early spore formation was also observed in this investigation. The three Fusarium species sporulated readily on the maize leaf surface; F. graminearum sporulated very early after inoculation (48 hai); F. proliferatum and F. verticillioides formed spores later (72 hai). Sporulation on the leaf surface was detected later, 9-18 dai. The late spore formation observed may have been due to fungal hyphae that re-emerged from the infected tissues. Interestingly, the spores were observed inside the leaf tissues and these spores were observed to be released through stomata and trichomes at 7 dai. However, this phenomenon was only observed in F. proliferatum inoculated onto maize leaves. The sporulation of other Fusarium spp. inside the leaves of other plants has been reported. For instance, Wagacha (2012) reported that F. tricinctum produced spores inside the leaves of wheat plants, while on the other hand, F. verticillioides spores were detected inside maize seedlings at 21 dai (Oren et al. 2003). The contrast between these results and those of Oren et al. (2003) may be explained by the type of cells infected (mesocotyl cells) and the inoculation method used. The phenomenon of secondary spore production that was witnessed in the present study constitutes a potential avenue for the preservation of genetic diversity during unexpected environmental conditions. The relation of sporulation of Fusarium species during early stages of growth to later periods of the infection process imply that the spores disseminate efficiently to upper leaves and to silks.

Acknowledgements

This work was funded by Plant and Food Biosecurity, 7th Framework Program, G.A. Nr. 261752. We gratefully acknowledge support from the Vietnamese Ministry of Education and Training and t h e G erman Academic Exchange Service (DAAD) for fellowship support. We thank Dr. Joachim Hamacher and Inge Neukirchen for performing the TEM. We thank Prof. Richard A. Sikora and Dr. Charles Howie for providing language help. 9

ACCEPTED MANUSCRIPT

References Bacon CW, Glenn AE, Yates IE, 2008. Fusarium verticillioides: managing the endophytic association with maize for reduced fumonisins accumulation. Toxin Review 27:411-446

RI PT

Bacon CW, Hinton DM, 1996. Symptomless endophytic colonization of maize by Fusarium moniliforme. Canadian Journal of Botany 74:1195-1202 Bacon CW, White J, 2000. Physiological adaptations in the evolution of endophytism in the Clavicipitaceae. Marcel Dekker. New York. pp:237-261

SC

Bai GH, Shaner G, 1996. Variation in Fusarium graminearum and cultivar resistance to wheat scab. Plant disease 80:975-979 Boenisch M, Schäfer W, 2011. Fusarium graminearum forms mycotoxin producing infection structures on wheat. BMC Plant Biology 11:110

M AN U

Bruzzese E, Hasan S, 1983. A whole leaf clearing and staining technique for host specificity studies of rust fungi. Plant Pathology 32:335-338 Burgess L, Dodman R, Pont W, Mayers P, 1981. Fusarium diseases of wheat, maize and grain sorghum in eastern Australia. In: Nelson PE, Toussoun TA, and Cook RJ (eds), Fusarium: diseases, biology and taxonomy. University Park, PA 16802:64-76 Canny M, 1995. Apoplastic water and solute movement: new rules for an old space. Annual Review of Plant Biology 46:215-36

TE D

Chungu C, Mather DE, Reid LM, Hamilton RI, 1996. Comparison of techniques for inoculating maize silk, kernel, and cob tissues with Fusarium graminearum. Plant Disease 80:81-84 Desjardins AE, Plattner RD, Lu M, Claflin LE, 1998. Distribution of fumonisins in maize ears infected with strains of Fusarium moniliforme that differ in fumonisin production. Plant Disease 82:953-958

EP

Ding L, Xu H, Yi H, Yang L, Kong Z, Zhang L, Xue S, Jia H, Ma Z, 2011. Resistance to hemibiotrophic F. graminearum infection is associated with coordinated and ordered expression of diverse defense signaling pathways. Plos One 6:1-17

AC C

Doohan FM, Brennan J, Cooke BM, 2003. Influence of climatic factors on Fusarium species pathogenic to cereals. European Journal of Plant Pathology 109:755-768 Dutton MF, 2009. The African fusarium/maize disease. Mycotoxin Research 25:29-39 Geyer

G, 1973. Ultrahistochemie: Histochemische Elektronenmikroskopie. Gustav Fischer Verlag Jena

Arbeitsvorschriften

für

die

Gilbertson R, Brown Jr W, Ruppel E, 1985. Prevalence and virulence of Fusarium spp. associated with stalk rot of corn in Colorado. Plant Disease 69:1065-1068 Gilbertson RL, 1986. Association of corn stalk rot Fusarium spp. and Western corn rootworm beetles in Colorado. Phytopathology 76:1309-1314

10

ACCEPTED MANUSCRIPT

Görtz A, Zuehlke S, Spiteller M, Steiner U, Dehne H, 2010. Fusarium species and mycotoxin profiles on commercial maize hybrids in Germany. European Journal of Plant Pathology 128:101-111

RI PT

Jansen C, Von Wettstein D, Schäfer W, Kogel KH, Felk A, Maier FJ, 2005. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum. Proceedings of the National Academy of Sciences of the United States of America 102:16892-16897 Kabeere F, Hampton JG, Hill MJ, 1997. Transmission of Fusarium graminearum (Schwabe) from maize seeds to seedlings. Seed Science and technology 25 (2):245-252.

SC

Kang Z, Buchenauer H, 2000a. Cytology and ultrastructure of the infection of wheat spikes by Fusarium culmorum. Mycological Research 104:1083-1093

M AN U

Kang Z, Buchenauer H, 2000b. Ultrastructural and cytochemical studies on cellulose, xylan and pectin degradation in wheat spikes infected by Fusarium culmorum. J. Phytopathol. 148:263-275 Karnovsky MJ, 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J. cell Biol 27:137-8A Klein T, Burgess L, Ellison F, 1991. The incidence and spatial patterns of wheat plants infected by Fusarium graminearum group 1 and the effect of crown rot on yield. Crop and Pasture Science 42:399-407

TE D

Knight NL, 2011. Quantitative PCR and histopathological assessment of cereal infection by Fusarium pseudograminearum. PhD thesis University of Southern Queensland, Australia. Koehler B, 1942. Natural mode of entrance of fungi into corn ears and some symptoms that indicate infection. Journal of Agricultural Research 64:421-442

EP

Lamprecht S, Marasas W, Alberts J, Cawood M, Gelderblom W, Shephard GS, Thiel PG, Calitz FJ, 1994. Phytotoxicity of fumonisins and TA-toxin to corn and tomato. Phytopathology 84:383-391

AC C

Larran S, Perelló A, Simón MR, Moreno V, 2007. The endophytic fungi from wheat (Triticum aestivum L.). World Journal of Microbiology and Biotechnology 23:565-572 Lawrence EB, Nelson PE, Ayers JE, 1981. Histopathology of sweet corn seed and plants infected with Fusarium moniliforme and F. oxysporum. Phytopathology 71:379-386 Logrieco A, Mule G, Moretti A, Bottalico A, 2002. Toxigenic Fusarium species and mycotoxins associated with maize ear rot in Europe. European Journal of Plant Pathology 108:597609 Mary Wanjiru W, Zhensheng K, Buchenauer H, 2002. Importance of cell wall degrading enzymes produced by Fusarium graminearum during infection of wheat heads. European Journal of Plant Pathology 108:803-810 Moradi GM, 2008. Microbiological and molecular assessment of interactions among the major Fusarium head blight pathogens on wheat ears. PhD thesis Universität Bonn, Germany 11

ACCEPTED MANUSCRIPT

Munkvold GP, 2003. Epidemiology of Fusarium diseases and their mycotoxins in maize ears. European Journal of Plant Pathology 109:705-713 Munkvold GP, Desjardins AE, 1997. Fumonisins in maize: Can we reduce their occurrence? Plant Disease 81:556-65

RI PT

Munkvold GP, Hellmich RL, Showers WB, 1997a. Reduced Fusarium Ear Rot and symptomless infection in kernels of maize genetically engineered for European corn borer resistance. Phytopathology 87:1071-1077 Munkvold GP, McGee DC, Carlton WM, 1997b. Importance of different pathways for maize kernel infection by Fusarium moniliforme. Phytopathology 87:209-217

SC

Murillo I, Cavallarin L, Segundo BS, 1999. Cytology of infection of maize seedlings by Fusarium moniliforme and immunolocalization of the pathogenesis-related PRms protein. Phytopathology 89:737-747

M AN U

Ochor T, Trevathan L, King S, 1987. Relationship of harvest date and host genotype to infection of maize kernels by Fusarium moniliforme. Plant Disease 71:311-313 Oiah B, Jeney A, Hornok L, 2006. Transient endophytic colonization of maize tissues by Fusarium proliferatum. Acta Phytopathologica et Entomologica Hungarica 41:185-191 Oren L, Ezrati S, Cohen D, Sharon A, 2003. Early events in the Fusarium verticillioides-maize interaction characterized by using a green fluorescent protein-expressing transgenic isolate. Appl. Environ. Microbiol. 69:1695-1701

TE D

Parry D, Jenkinson P, McLeod L, 1995. Fusarium ear blight (scab) in small grain cereals—a review. Plant Pathology 44:207-238 Pastirčák M, 2004. The Effect of conidial suspension of fungi Fusarium graminearum and F. moniliforme on maize seedling growth. Acta fytotechnica et zootechnica, 7:231-233

EP

Pritsch C, Muehlbauer GJ, Bushnell WR, Somers DA, Vance CP. 2000. Fungal development and induction of defense response genes during early infection of wheat spikes by Fusarium graminearum. Molecular Plant-Microbe Interactions 13:159-69

AC C

Rahman MME, Ali ME, Ali MS, Rahman MM, Islam MN, 2008. Hot water thermal treatment for controlling seed-borne mycoflora of maize. Crop Production 3(5): 5-9 Reid LM, 1992. Genotypic differences in the resistance of maize silk to Fusarium graminearum. Canadian Journal of Plant Pathology 14:211-213 Reid LM, Woldemariam T, Zhu X, Stewart DW, Schaafsma AW, 2002. Effect of inoculation time and point of entry on disease severity in Fusarium graminearum, Fusarium verticillioides, or Fusarium subglutinans inoculated maize ears. Canadian Journal of Plant Pathology 24: 162-167 Santiago R, Reid LM, Arnason JT, Zhu X, Martinez N, Malvar RA, 2007. Phenolics in maize genotypes differing in susceptibility to Gibberella Stalk Rot (Fusarium graminearum Schwabe). Journal of Agricultural and Food Chemistry 55:5186-5193 Schulz B, Boyle C, 2005. The endophytic continuum. Mycological Research 109:661-686 12

ACCEPTED MANUSCRIPT

Schulz B, Römmert A-K, Dammann U, Aust H-J, Strack D, 1999. The endophyte-host interaction: a balanced antagonism? Mycological Research 103:1275-1283 Southwell R, Moore K, Manning W, Hayman P, 2003. An outbreak of Fusarium head blight of durum wheat on the Liverpool Plains in northern New South Wales in 1999. Australasian Plant Pathology 32:465-471

RI PT

Spurr AR, 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research 26:31-43 Sutton JC, 1982. Epidemiology of wheat head blight and maize ear rot caused by Fusarium graminearum. Canadian Journal of Plant Pathology 4:195- 209

SC

Tejera N, Ortega E, Rodes R, Lluch C, 2006. Nitrogen compounds in the apoplastic sap of sugarcane stem: Some implications in the association with endophytes. Journal of Plant Physiology 163:80-85

M AN U

Thomas MD, 1980. Incidence and persistence of Fusarium moniliforme in symptomless maize kernels and seedlings in Nigeria. Mycologia 72:882-887 Trail F, 2009. For blighted waves of grain: Fusarium graminearum in the postgenomics era. Plant Physiology 149:103-110 Vieira MLC, 2000. Symptomless infection of banana and maize by endophytic fungi impairs photosynthetic efficiency. The New Phytologist 147:609-615

TE D

Wagacha JM, Oerke E-C, Dehne H-W, Steiner U, 2012. Colonization of wheat seedling leaves by Fusarium species as observed in growth chambers: a role as inoculum for head blight infection? Fungal Ecology 5 (5): 581-590 Williams LD, Glenn AE, Zimeri AM, Bacon CW, Smith MA, Riley RT, 2007. Fumonisin disruption of ceramide biosynthesis in maize roots and the effects on plant development and Fusarium verticillioides-induced seedling disease. J. Agric. Food Chem. 55(9): 2937-2946

EP

Yates IE, Bacon C, Hinton D, 1997. Effects of endophytic infection by Fusarium moniliforme on corn growth and cellular morphology. Plant Disease 81:723-728

AC C

Yates IE, Hiett KL, Kapczynski DR, Smart W, Glenn AE, Hinton DM, Bacon CW, Meinersmann R, Liu S, Jaworski AJ, 1999. GUS transformation of the maize fungal endophyte Fusarium moniliforme. Mycological Research 103:129-136

13

ACCEPTED MANUSCRIPT

a

20µm

d

20µm

b

20µm

c

20µm

M AN U

SC

20µm

RI PT

A

A

e

20µm

f

AC C

EP

TE D

Figure 1. Infection of asymptomatic maize leaves by Fusarium species. a: hyphal swelling (A) occuring over infected sites. b: hypha within epidermal cell and swelling at a hyphal tip formation at crossing wall. c: hyphal growth within epidermal cell. d and e: hyphal growth as a finger‐shaped subcuticular structure. f: intercellular hyphal growth. a ‐ c: F. graminearum. d and e: F. proliferatum. f: F. verticillioides. a. Arrows indicate the hyphae on or in the cells. Light micrographs.

ACCEPTED MANUSCRIPT

H

20µm

20µm

b

20µm

c

M AN U

SC

a

RI PT

IH

IH H

AH

IH

20µm

20µm

e

20µm

f

TE D

d

TH

IH

AH

2.5µm

EP

AC C

IH

IH

g

1 µm

CW

h

0.5µm

k

Figure 2. Infection of symptomatic maize leaves by Fusarium species. a: subcuticular hyphae (H) growing

along cell wall of bulliform cell. b: hyphae invading subcuticle of short epidermal cell (black arrow) and spreading into adjacent cell (white arrow). c, d and g: Intercellular hyphae r (IH) in a necrotic lesion (heavy symptom leaf). i: Hyphae invading intercellular (IH) spaces in a mild symptomatic leaf. e and h: Hyphae growing in mesophyl cells, intracellular hyphae (AH) and thickened hyphae (TH) in a necrotic lesion (heavy symptom leaf). a‐c: F. graminearum. d, e, g and h: F. proliferatum. f and i: F. verticillioides. a‐f: light micrographs. g‐k: Transmission electron micrographs.

ACCEPTED MANUSCRIPT

b

DP

20µm

20µm

IC

e

20µm

f

A

i

TE D

A

10µm

RI PT

20µm

c

20µm

d

SC

a

M AN U

20µm

20µm

IP

IC

A

j

20µm

10µm

g

20µm

h

s

k

20µm

l

AC C

EP

Figure 3. Infection of stomata of maize leaves by Fusarium species. a, i and j: hyphal swelling at a tip (A), occurring at stomatal aperture. e: Direct penetration (DP). b and f infection cushion (IC) formation on surface of stomata. c: imprint circles (IP) below infection cushion. d: hyphae in sub‐stomatal cavity. g and l: hyphae in sub‐stomatal cavity and spreading to adjacent cells. e: hyphae penetrating directly through stomatal aperture. h: hyphae re‐emergence from stoma at edge lesion. k: hyphae spreading to subcuticle. a ‐ d: F. graminearum. e ‐h : F. proliferatum. I‐ l: F. verticillioides. Arrows indicate hyphae on or in cells. Light micrographs.

ACCEPTED MANUSCRIPT

IC

RI PT

S

M AN U

SC

10µm

2.5µm

1

2

3

c

2.5µm

EP

TE D

b

a

d

2.5µm

e

2.5µm

f

AC C

Figure 4. Stomata and infection via stomata of maize leaves by Fusarium proliferatum. a: Stomata in control treatment (inoculated with water). b: Infection cushion (IC) formation on stomatal surface (S), penetration through stomatal aperture and growth in substomatal cavity (black arrow) and intercellular space (white arrow). c: close –up infected stomata in a. d: box area 1 in b, close ‐ up infected cushion. e: close ‐ up box area 2 in b, hypha penetrating through stomatal aperture. f: close ‐ up box area 3 in b, hypha enlarging and branching in substomatal cavity. a SEM photographs, b vertical section, Light micrograph. b‐e: Transmission electron microsgraphs.

ACCEPTED MANUSCRIPT

F. graminearum

F. proliferatum

F. verticillioides a

RI PT

350 300 250 200 a

150

b

100 50

b b

7 dai

b

15 dai

M AN U

0

SC

Infected stomata/cm2

400

AC C

EP

TE D

Figure 5. Infection via stomata by Fusarium species on maize leaves at 7 and 15 dai. For each sampling time, mean values followed by the same letters are not significantly different P≤ 0.05 (Duncan’s test). Error bars represent the standard error of the mean.

a

20µm

b

20µm

c

20µm

d

e

1µm

f

TE D

50µm

M AN U

SC

50µm

RI PT

ACCEPTED MANUSCRIPT

20µm

g

20µm

h

20µm

k

20µm

l

T

i

20µm

EP

20µm

j

AC C

Figure 6. Sporulation of Fusarium species on the maize leaf. a: conidiaphore produced from re‐ emergence hyphae. b: conidiophores produced from hyphae on leaf surface. c and d: conidiophore and spore formed from macroconidia. e: macroconidium forming on leaf surface by secondary hyphae. f: Conidia formed inside destroyed leaf tissue. g: microconidia formed in substomatal cavity. h: microconidia sporulating through stomata. i: phialides forming from hyphae re‐ emerging from necrotic lesion. J: microconidiaphore forming from hyphae re‐ emerging from trichome (T). k: microconidia producing from germ tube and sporulating. L: microconidia producing from aerial hypha. a‐d: Fusarium graminearum, e ‐ J: F. proliferatum. k and l: F. verticillioides. Arrows indicate the conidia on or in the tissue. a‐e and g‐l: Light micrographs. f: Transmission electron micrographs.