Both authors contributed equally.
Bipolaris sorokiniana, a cereal pathogen of global concern: cytological and molecular approaches towards better control‡
Article first published online: 3 JUL 2002
Molecular Plant Pathology
Volume 3, Issue 4, pages 185–195, July 2002
How to Cite
Kumar, J., Schäfer, P., Hückelhoven, R., Langen, G., Baltruschat, H., Stein, E., Nagarajan, S. and Kogel, K.-H. (2002), Bipolaris sorokiniana, a cereal pathogen of global concern: cytological and molecular approaches towards better control. Molecular Plant Pathology, 3: 185–195. doi: 10.1046/j.1364-3703.2002.00120.x
Additional cytological images from Bipolaris –cereal interactions under: http://www.uni-giessen.de/ipaz .
- Issue published online: 3 JUL 2002
- Article first published online: 3 JUL 2002
Bipolaris sorokiniana (teleomorph Cochliobolus sativus ) is the causal agent of common root rot, leaf spot disease, seedling blight, head blight, and black point of wheat and barley. The fungus is one of the most serious foliar disease constraints for both crops in warmer growing areas and causes significant yield losses. High temperature and high relative humidity favour the outbreak of the disease, in particular in South Asia's intensive ‘irrigated wheat–rice’ production systems. In this article, we review the taxonomy and worldwide distribution, as well as strategies to counteract the disease as an emerging threat to cereal production systems. We also review the current understanding of the cytological and molecular aspects of the interaction of the fungus with its cereal hosts, which makes B. sorokiniana a model organism for studying plant defence responses to hemibiotrophic pathogens. The contrasting roles of cell death and H 2O2 generation in plant defence during biotrophic and necrotrophic fungal growth phases are discussed.
A detailed description of Bipolaris sorokiniana (Sacc.) Shoemaker (Sivanesan, 1990) can be obtained from C.M.I.'s Sivanesan and Holliday (1981). In the older literature, several synonyms of the anamorph have been used: Helminthosporium sorokinianum, Drechslera sorokiniana, and Helminthosporium sativum (Maraite et al., 1998). Shoemaker (1959) proposed the generic name Bipolaris for the Helminthosporium species with fusoid, straight, or curved conidia germinating by one germ tube from each end (bipolar germination, Fig. 1A). (The former genus Helminthosporium was divided into three anamorphic genera: Bipolaris, Drechslera, and Exserohilum with the teleomorphic stages Cochliobolus, Pyrenophora, and Setosphaeria, respectively.) B. sorokiniana is characterized by thick-walled, elliptical conidia (60–120 µm × 12–20 µm) with five to nine cells. In axenic culture, the mycelium is composed of hyphae interwoven as a loose cottony mass and appears white or light to dark grey depending on the isolates (Fig. 1B). The fungus is differentiated from other members of the Bipolaris genus on the basis of morphological features of conidiophores and conidiospores. A key for distinguishing species of Bipolaris was described by Subramanian (1971).
The ascigerous state was observed in the laboratory on natural media in the presence of opposite mating types, and was first described as Ophiobolus sativus. It was later renamed Cochliobolus sativus (Ito & Kuribayashi) Drechsler ex. Dastur, 1942 (Dastur, 1942). Under natural conditions, the perfect stage was only found in Zambia (Raemaekers, 1988), and it has not been reported to occur in any other areas in which the pathogen prevails. The fungus belongs to the subdivision Ascomycotina (class: Loculoascomycetes; order: Pleosporales; family: Pleosporaceae). The genus Cochliobolus is characterized by globose ascomata with a long cylindrical neck, obclavate cylindrical asci, and helically coiled filiform ascospores. It is associated with Bipolaris and Curvularia anamorphs.
The host range
B. sorokiniana affects small grain cereals, although rye is less susceptible and oats are seldom infected ( Zillinsky, 1983 ). A wide variety of other grasses act as potential hosts. Table 1 summarizes, as an example, the host range on monocotyledonous plants evaluated in western Hungary ( Bakonyi et al., 1997 ).
B. sorokiniana causes foliar spot blotch, root rot, and black point on grains ( Fig. 2A–G ), as well as head blight and seedling blight of wheat and barley. Infected seedlings develop dark brown necrotic lesions on roots, crowns, and lower leaf sheaths. Leaf blade and sheath infections develop as distinct oval to elongated light to dark brown blotches ( Fig. 2A–C ). Root ( Fig. 2D ) and crown ( Fig. 2E ) infections can be so severe that infected plants dry out without producing any seed. Under favourable conditions, spikelets may be affected, causing grain shrivelling ( Fig. 2F,G ). The susceptibility to the pathogen increases around Zadoks’ growth stage DC 56 (three-quarters of the inflorescence emerged). At this stage, if weather conditions are conducive, i.e. continuous rain for 5–6 days followed by warmer temperatures (day average of 20–30 °C), spot blotch epidemic can develop very rapidly ( Mehta, 1998 ). Under low light intensity, the pathogen may colonize host tissue intercellularly without causing visible damage. Fungal infection accelerates leaf senescence at later growth stages ( Dehne and Oerke, 1985 ).
Variability in fungal populations
Physiological specialization at the species level was first described by Christensen (1926), who showed that fungal isolates varied considerably in virulence to wheat and barley. Differential reactions of progenies of crosses between isolates that differed in pathogenicity to different grass species indicated complex inheritance involving several genes (Nelson, 1960, 1961). Specificity on the race-cultivar level is indicated by the observation that field populations shift to more aggressive races with long-term continuous wheat cultivation (El-Nashaar and Stack, 1989). Valjavec-Gratian and Steffenson (1997a,b) identified three pathotypes from 33 isolates of North Dakota, tested on the basis of interaction phenotypes with three six-rowed barley differentials. There are several additional reports on the genetic variability of B. sorokiniana populations (Fetch and Steffenson, 1994; Misra, 1979). Recent studies based on large numbers of strains collected on a global basis suggest that B. sorokiniana forms a continuum of isolates varying in virulence and aggressiveness with specific and nonspecific interactions (Duveiller and Garcia Altamirano, 2000; Maraite et al., 1998). The mechanism of variability is not well understood. Fusions (anastomosis) between hyphae, that stem from different conidia, may result in somatic hybridization and the emergence of new fungal variants (Fig. 1F).
Worldwide distribution of the pathogen
Owing to the increasing global demand for wheat-based products, wheat cultivation, which was earlier confined to rain-fed situations, has extended to irrigated systems (Dubin and Rajaram, 1996). Breeding efforts and irrigation have increased cropping intensity, thereby supporting the spread of the pathogen. Since significant progress has been obtained in the resistance to leaf rust, resulting in the absence of a major leaf rust epidemic in recent years, spot blotch has become a major production constraint in South Asia's intensive cropping systems, where nearly 12 000 000 ha are affected (Nagarajan and Kumar, 1998; Ruckstuhl, 1998; Singh et al., 1998). Due to replacements of land-race varieties by high-yielding, rust-resistant genotypes, wheat production is also threatened in parts of China (Chang and Wu, 1998). The pathogen also occurs in North and Latin America (Duczek and Jones-Flory, 1994), Brazil (Mehta et al., 1992), and, much less frequently, in parts of Europe (Kwasna, 1995) (Fig. 3).
Disease resistance and control
Host responses in spot blotch disease
As a hemibiotrophic pathogen, B. sorokiniana exerts a biotrophic and a subsequent necrotrophic growth phase. Microscopic analysis of fungal development in leaves of a susceptible barley (cv. Ingrid) has clearly shown the distinct successive growth phases characterized by: (i) cuticle and cell wall penetration, followed by the development of hyphae within the invaded, living epidermal host cell (biotrophic phase, Fig. 1C); and (ii) hyphae invasion into the mesophyll layer, accompanied by epidermal and mesophyll cell death (necrotrophic phase, Fig. 1D; additional cytological images under http://www.uni-giessen.de/ipaz). In rare cases, the pathogen penetrates via stomata (Fig. 1E). Host cell collapse in the necrotrophic phase is also indicated by an increase in electrolyte leakage coinciding with disease development (Wisniewska et al., 1998). Cell collapse is brought about by toxin secretion because cells die without direct contact with fungal hyphae, and toxin infiltration into leaves elicits an indistinguishable necrotic host response (Kumar et al., 2001). Epifluorescence microscopy (Fig. 4A,C,E), in combination with the use of the fluorescent brightener calcofluor (Fig. 4B,D,F), shows considerable potential for studying the interaction of the pathogen with host cells. Calcofluor has been used for cell and hyphal wall detection (Rohringer et al., 1977) and facilitates the location of fungal penetration sites in leaf tissue. Papilla-like structures with a halo appear light blue, probably indicating cell wall alterations by the activity of fungal hydrolases.
Histochemical studies using 3,3-diaminobenzidine (DAB) to visualize the reactive oxygen intermediate hydrogen peroxide (H2O2) show the association of fungal development with an oxidative burst in epidermal and mesophyll cells (Fig. 5; Kumar et al., 2001). A positive correlation between host susceptibility (establishment of spot blotch) and the amount of H2O2 produced in leaf lesions suggests that H2O2 is involved in successful tissue infection (Fig. 5A). This hypothesis is in agreement with earlier reports that demonstrated the association of successful growth of the necrotrophic pathogens Botrytis cinerea and Sclerotium sclerotiorum with H2O2 production by the host plant (Govrin and Levine, 2000). Notably, the early biotrophic growth phase in barley, which is confined to single-cell interaction of the fungal hypha with an epidermal cell, is also accompanied by H2O2 production, although this burst is confined to papillae and anticlinal cell walls at the sites of fungal attack (Fig. 5B–D). In some cases, brownish DAB staining overspreads attached epidermal cells (Fig. 5E) followed by rapid cell death. Based on many microscopic data, there is correlative evidence that these early host responses are associated with fungal growth retardation. Consistently, cell invasion and successful single-cell interaction are not associated with DAB staining (Fig. 5F). These observations suggest that H2O2 plays an ambivalent role in the host, depending on its spatiotemporal accumulation profile (Kumar et al., 2001). Accumulation of hydrogen peroxide, papilla formation, and hypersensitive cell death are well-known early plant defence responses to biotrophic pathogens, such as Blumeria graminis (Görlach et al., 1996; Hückelhoven et al., 1999; Thordal-Christensen et al., 1997). Hence, at early interaction stages, barley seems to exert the same effective defence features against hemibiotrophic and biotrophic pathogens.
Upon inoculation of rice with B. sorokiniana, pathogenesis-related transcripts PR-1, PR-2, PR-3, PR-4, PR-5, as well as peroxidase, accumulate by 12 h (Manandhar et al., 1999). Infiltration of culture filtrate into barley first leaves induces transcripts of PR-1 as well as BAX Inhibitor-1 and aspartate protease (R. Hückelhoven, unpublished results), which are typically expressed during the hypersensitive response to the biotrophic powdery mildew fungus (Hückelhoven et al., 2001). Phenylalanine ammonia-lyase (PAL) activity is strongly induced in barley and wheat leaves after inoculation with different fungal isolates. Aggressive ones seem to generate stronger and early PAL induction, which might suggest that activation of the phenylpropanoid pathway is not associated with defence to B. sorokiniana. Gene-specific probes were used to assess the expression patterns of four different PAL clones (hpa12, hpa13, hpa14, and hpa16) in barley leaves and cell cultures (Kervinen et al., 1998). The genes were all pathogen-responsive, although with considerable variation in their expression level and timing.
There are many early reports on the presence of phytotoxic substances in cultural filtrates of B. sorokiniana (Gayad, 1961; Ludwig, 1957). The fungus produces sesquiterpenoid toxins that are synthesized from farnesol. About 20 compounds related to helminthosporol have been isolated from different species of the genus Bipolaris (Kachlicki, 1995; Turner and Aldridge, 1983). Prehelminthosporol (Fig. 6A) is the most abundant and active compound of B. sorokiniana; it is produced in amounts of 1.2–2.1 µg/mg dry matter of fungal tissue (Carlson et al., 1991). Although the prehelminthosporol sensitivity of barley cultivars is not directly correlated with levels of disease resistance, it is supposed to play a role in pathogenesis by killing or weakening plant cells (Liljeroth et al., 1993). Its aldehyde form, prehelminthosporal, exerts an inhibitory effect on proton pumping by H+-ATPase in a dose-dependent manner (Olbe et al., 1995). Furthermore, it appears to inhibit enzyme activities of Ca2+-ATPase and β-1,3-glucan synthase (callose synthase). Helminthosporol (Fig. 6B) affects membrane permeability, thereby inhibiting mitochondrial oxidative phosphorylation, the photophosphorylation in chloroplasts, and the proton pumping across the plasma membrane, as well as β-1,3-glucan synthase activity (Briquet et al., 1998). The data suggest that the toxins of the helminthosporol family act like classical uncouplers rather than via a specific interaction with a host membrane receptor.
A low-temperature preparation technique, in combination with immunogold labelling, was used for the localization of prehelminthosporol in hyphae and germinated conidia. Major labelling was seen in membrane-bound organelles identified as Woronin bodies. These spherical, hexagonal, or rectangular proteinaceous crystals plug the septal pores, thereby distinguishing old damaged fungal hyphae from the rest of the mycelium (Akesson et al., 1996).
Recently, a compound called ‘sorokinianin’ (Fig. 6C) has been isolated from fungal culture filtrates (Nakajima et al., 1994). The compound shows inhibitory activity on barley seed germination. Using two-dimensional nuclear magnetic resonance (2D-NMR) analysis, it was demonstrated that the compound is a condensation product originating from products of the sesquiterpene and TCA pathways (Nakajima et al., 1998).
Cellulose-hydrolysing enzymes from B. sorokiniana have been partially purified and characterized (Geimba et al., 1999). β-Glucosidase and cellobiohydrolase activities were higher on cellulose-containing media, indicating substrate activation. A fungal 30 kDa endo-1,4-β-xylanase was isolated from infected roots and stems and was serologically related to xylanase produced by Pyrenophora teres and P. graminea (Peltonen, 1995).
Sources of disease resistance
The host response of segregating populations is best distinguished on the basis of their response measured as the area under the disease progress curve (AUDPC) (Duveiller et al., 1998; Sharma et al., 1997). A low AUDPC was associated with a higher biomass and grain yield, higher harvest index, and a higher 1000—kernel weight. Alternatively, ergosterol has been used as a biochemical marker to detect and quantify fungal mycelium in leaves (Gunnarsson et al., 1996). However, a conclusive correlation could not be seen between disease level and ergosterol content. However, there was a good correlation between ergosterol content and GUS activity, as well as between GUS activity and lesion size, in barley roots infected with a GUS-transformed strain of B. sorokiniana (Liljeroth et al., 1996), suggesting that this method might provide an effective tool for reliable disease evaluation under laboratory practice.
Genetics of host resistance
Integrated strategies for controlling B. sorokiniana on wheat and barley include resistance breeding, chemical control, soil and residue management, and crop rotation (Mehta, 1998). In general, the degree of resistance in modern cultivars is still insufficient (Chang et al., 1998; Hetzler et al., 1991; Mujeeb-Kazi, 1998; Van Ginkel and Rajaram, 1998). Progress was made by transferring resistance genes from Thinopyrum curvifolium, Elymus curvifolius, and Triticum tauschii to wheat (Mujeeb-Kazi et al., 1996a,b). Resistance to common root rot was obtained after crossing Aegilops ovata with Triticum aestivum using Chinese Spring phIb genetic stock and the cultivar ‘Leader’. This germplasm represents a different source of genes for resistance to common root rot than generally available in spring wheat cultivars (Bailey et al., 1995). Grain yield increased in cultivars with resistance traits derived from Thinopyrum curvifolium and Chinese germplasm. This germplasm has been identified as a valuable source of genes for spot blotch resistance in wheat (Sharma et al., 1997; Villareal et al., 1995).
Spot blotch resistance is governed by quantitative traits. Two quantitative trait loci (QTLs) mapped to chromosome 1S and 5S, respectively, have been identified in barley for spot blotch resistance at the adult stage (Steffenson et al., 1996). There is one report on a monogenically inherited resistance trait that is observed only at the barley seedling stage (Valjavec-Gratian and Steffenson, 1997a). The locus mapped to the distal region of chromosome 1S near to the above-mentioned QTL. Kutcher et al. (1994) analysed the heritability of resistance to common root rot and spot blotch in barley. The heritability of root rot resistance of Cross Fr926-77 × Deuce ranged from 56 to 85%, whilst the heritability for spot blotch resistance in the same cross was 43–61%. Thus, different traits seem to influence host response in barley roots and leaves.
Effects of the barley Mlo locus on spot blotch development
In an attempt to identify new durable disease resistance traits in cereals that control spot blotch, the influence of the barley Mlo locus on disease development has been evaluated. This locus is known to control very efficiently race-nonspecific resistance to biotrophic Blumeria graminis in a recessive manner, explaining why more than 70% of European barley genotypes contain mlo resistance alleles (Jørgensen, 1994). Strikingly, all tested Blumeria graminis-resistant barley genotypes (mlo lines) were hypersusceptible to B. sorokiniana culture filtrates when compared with the wild-type (Mlo lines) (Kumar et al., 2001). The same ambivalent effect of the Mlo locus was seen in the interaction of barley with the hemibiotrophic fungus Magnaporthe grisea (Jarosch et al., 1999). Hypersusceptibility of mlo genotypes can be interpreted on the basis of recent progress in the understanding of the molecular role of the Mlo gene in powdery mildew resistance. The wild-type product, MLO, is required for the establishment of a compatible interaction of barley and the powdery mildew fungus, as mutants lacking MLO (mlo lines) are highly resistant to this fungus. Suspiciously, mlo mutants undergo spontaneous cell death in fully developed green leaves under aseptic conditions (Peterhänsel et al., 1997). It was therefore suggested that MLO is a negative control element of cell death (Büschges et al., 1997). Since the survival of a hemibiotrophic pathogen in its necrotrophic phase depends on host cell death as a prerequisite for successful pathogenesis, breakdown of cell survival mechanisms as a result of mutations in the Mlo gene may antagonize plant defence to B. sorokiniana and M. grisea.
Compared to wild-type barley, mlo mutants accumulate more H2O2 and develop leaf necrosis in response to toxic culture filtrates of B. sorokiniana (Kumar et al., 2001). Recently, we found a link between MLO and the oxidative burst via a putative NADPH oxidase activator protein RAC (Schultheiss et al., 2002). Due to transient Rac gene silencing as a result of sequence-specific RNA interference induced by RacB-dsRNA in wild-type barley (Mlo lines), penetration rates of Blumeria graminis are drastically reduced. This resistance-inducing effect of RacB-dsRNA, like mlo resistance, depends on the function of another barley gene Ror1 (required for mlo-specified resistance; Schultheiss et al., 2002). Like MLO, RAC seems to play an ambivalent role in the resistance of grasses to biotrophic and hemibiotrophic fungi. The over-expression of active RAC in rice plants enhances resistance to M. grisea, whereas the dominant negative mutant RAC provokes enhanced susceptibility (Ono et al., 2001). These intriguing observations suggest that MLO, together or in parallel with RAC, plays a significant role in affecting hemibiotrophic pathogens. The early production of superoxide anion radicals mediated by RAC via interaction with NADPH oxidase may therefore limit the development of B. sorokiniana through cell wall reinforcement and hypersensitivity as well as antioxidant induction.
Induced resistance and biological control
On a worldwide scale, yield losses in wheat and barley caused by B. sorokiniana indicate the need to search for alternative strategies for disease control. One of the promising strategies is induced resistance. In the broadest sense, induced resistance means the control of parasite and pest by a prior activation of the plant's own defence system. Defence is activated by necrotizing pathogens as well as by chemicals mimicking factors of the natural defence systems, such as salicylic acid (Bochow et al., 2001; Sticher et al., 1997). Chemical induction of resistance to B. sorokiniana in barley by pretreatment with the resistance inducers 2,6-dichloroisonicotinic acid (DCINA), benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methylester (BTH) or jasmonates leads to symptom reduction in the range of 10–20% (J. Kumar and A. Ibeagha unpublished results). The efficacy of the chemicals is rather unpredictable and resistance strategies by chemical inducers are inapplicable for farmers so far.
Instead, biological protection strategies look more promising. Pretreatment of wheat leaves with the inappropriate B. oryzae (inducer organism) reduced spot blotch lesion numbers and sizes of B. sorokiniana on the same leaves (Sarhan et al., 1991). A preliminary study identified antifungal substances in apoplastic fluids of ‘induced’ plants that were able to prevent spore germination of B. sorokiniana in vitro (Chakraborty and Sinha, 1984). Reduced symptoms could also be achieved, although not to the full extent, after spraying with the bacterial biocontrol agent Pseudomonas chlororaphis, strain MA 342. Treated seeds could be stored dry for at least 2 years without losing the disease-suppressing effect of the bacterial treatment (Johnsson et al., 1998).
Plant growth-promoting fungal isolates of Phoma spp. from zoysiagrass (Zoysia sp.) rhizosphere suppressed B. sorokiniana due to the competitive root colonization (Shivanna et al., 1996). Successful antagonists against seed-borne B. sorokiniana were Chaetomium sp., Idriella bolleyi, and Gliocladium roseum (Knudsen et al., 1995). Suppression of soil-borne fungi, including B. sorokiniana, has also been observed in the presence of isothiocyanates released into soil by Brassica species (Kirkegaard et al., 1996; Sarwar et al., 1998).
Encouraging results have recently been obtained in our laboratory on the control of spot blotch and root rot by Piriformospora indica, a plant growth-promoting root endophytic basidiomycete. Infestation of wheat roots by this fungus results in a considerable increase in growth and yield relative to non-infested controls. Shoot and root length, biomass, basal leaf area, overall size, and seed production were enhanced in the presence of the fungus by approximately 35%. Most intriguingly, the number and size of B. sorokiniana leaf lesions were significantly reduced (Table 2, as well as H. Baltruschat and P. Franken, unpublished results). These observations underline the value of biological strategies in crop protection systems. These strategies must be combined with appropriate plant production measures. In Canada, where common root rot prevails, rotation, including two or more years of flax (Linum usitatissimum) as a break crop, reduced the amount of viable inoculum of B. sorokiniana in the soil (Conner et al., 1996). Mixtures of wheat cultivars with different levels of resistance to spot blotch are a further option (Sharma and Dubin, 1996). Avoidance of the economically eligible zero tillage and stubble retaining is an additional measure that supports the control of crown rot and common root rot disease by farmers (Bailey and Duczek, 1996; Wildermuth et al., 1997).
|Treatment||Wheat* shoot fresh weight (g)†||Infection (% of leaf area)|
This work was supported by Alexander-von-Humboldt Stiftung, Bonn, Germany, Deutsche Volkswagen Stiftung, Wolfsburg, Germany, and Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany. We thank Dr Etienne Duveiller, Kathmandu, for critical discussions.
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