Evaluating the importance of the tan spot ToxA–Tsn1 interaction in Australian wheat varieties

The necrotrophic fungal pathogen Pyrenophora tritici‐repentis (Ptr) causes the major wheat disease tan spot, and produces multiple necrotrophic effectors that contribute to virulence. The proteinaceous effector ToxA induces necrosis in wheat genotypes possessing the Tsn1 gene, although the importance of the ToxA–Tsn1 interaction itself in varietal disease development has not been well studied. Here, 40 Australian spring wheat varieties were assessed for ToxA sensitivity and disease response to a race 1 wildtype Ptr isolate and ToxA‐deleted strain at both seedling and tillering growth stages. ToxA sensitivity was generally associated with disease susceptibility, but did not always predict spreading necrotic symptoms. Whilst the majority of Tsn1 varieties exhibited lower disease scores following toxa mutant infection, several exhibited no distinct differences between wildtype and toxa symptoms. This implies that ToxA is not the major determinant in tan spot disease development in some host backgrounds and indicates the presence of additional effectors. Unexpectedly, several tsn1 varieties exhibited a reduction in disease severity following toxa mutant inoculation, which may suggest an indirect role for ToxA in pathogen fitness. Additionally, increased chlorosis was observed following toxa mutant infection in three varieties, and further work is required to determine whether this is likely to be due to ToxA epistasis of ToxC symptoms. Taken together, these observations demonstrate that Ptr interacts with the host in a complex and intricate manner, leading to a variety of disease reactions that are dependent or independent of the ToxA–Tsn1 interaction.     

Revue bibliographique: la maladie de la tache auréolée du blé

Tan spot caused by Pyrenophora tritici‐repentis is a wheat disease found worldwide which can cause significant losses. This disease is characterized by typical symptoms: a necrotic spot surrounded by chlorosis halo. On the basis of its ability to produce chlorosis and/or necrosis symptoms on a differential host set. Eight races of this pathogen are currently recognized. These symptoms are the result of a specific interaction between the host and at least three host specific toxins Ptr ToxA, Ptr ToxB and Ptr ToxC. This interaction seems to be a mirror image of the classical gene‐for‐gene described by Flore. This paper presents a first literature review in the French language, identifying the major aspects of this disease, its epidemiology and diversity of its causal agent.     

Advances in the Characterization of the Pyrenophora tritici-repentis-Wheat Interaction

ABSTRACT Tan spot of wheat, caused by the fungus Pyrenophora tritici-repentis, is a destructive disease found in wheat-growing regions worldwide that can lead to serious yield losses. Changes in cultural practices have led to an increase in the severity and incidence of tan spot. Following infection, compatible races of the fungus elicit two distinct symptoms in differential wheat lines: tan necrosis and (extensive) chlorosis. Tan necrosis has been clearly demonstrated by several groups to result from the action of a protein toxin, Ptr ToxA. Wheat sensitivity to this toxin is conditioned by a single dominant gene. The chlorosis response may be more complex and appears to involve at least two other toxins, Ptr ToxB and Ptr ToxC, produced by different races of the fungus. Distinct genes apparently condition the reaction of wheat lines to each of these chlorosis-inducing toxins. This review concentrates on significant advances that have occurred during the past decade in the characterization of this disease interaction, ranging from the epidemiology and management of tan spot to molecular host-parasite interactions. Particular emphasis is placed on work describing fungal race differentiation, production of toxins and their importance in pathogenicity, and the genetics and physiology of host response to infection.     

Infection strategy of Ramularia collo‐cygni and development of ramularia leaf spot on barley and alternative graminaceous hosts

Ramularia leaf spot (RLS) is a newly important disease of barley across temperate regions worldwide. Despite this recent change in importance, the infection biology of the causal agent Ramularia collo‐cygni (Rcc) remains poorly understood. Confocal microscopy of the infection process of two transgenic Rcc isolates, expressing either GFP or DsRed reporter markers, was combined with light microscopy during field infection to track the progression of Rcc in planta. Infection of stomata, including the development of a previously unreported stomatopodium structure, results in symptomless development and intercellular colonization of the mesophyll tissue. Transition to necrotrophy is associated with breakdown of host chloroplasts and the formation of aggregates of conidiophores. In addition to barley, Rcc forms a compatible interaction with winter wheat and a number of perennial grass species. An incompatible reaction was observed with two dicotyledonous species. These results provide further insights into the host interactions of this fungus and suggest that RLS could be a potential threat to other agriculturally important crops.     

Effect of cold‐induced changes in physical and chemical leaf properties on the resistance of winter triticale (×Triticosecale) to the fungal pathogen Microdochium nivale

This study showed that several mechanisms of the basal resistance of winter triticale to Microdochium nivale are cultivar‐dependent and can be induced specifically during plant hardening. Experiments and microscopic observations were conducted on triticale cvs Hewo (able to develop resistance after cold treatment) and Magnat (susceptible to infection despite hardening). In cv. Hewo, cold hardening altered the physical and chemical properties of the leaf surface and prevented both adhesion of M. nivale hyphae to the leaves and direct penetration of the epidermis. Cold‐induced submicron‐ and micron‐scale roughness on the leaf epidermis resulted in superhydrophobicity, restricting fungal adhesion and growth, while the lower permeability and altered chemical composition of the host cell wall protected against tissue digestion by the fungus. The fungal strategy to access the nutrient resources of resistant hosts is the penetration of leaf tissues through stomata, followed by biotrophic intercellular growth of individual hyphae and the formation of haustoria‐like structures within mesophyll cells. In contrast, a destructive necrotrophic fungal lifestyle occurs in susceptible seedlings, despite cold hardening of the plants, with the host epidermis, mesophyll and vascular tissues being digested and becoming disorganized as a result of the low chemical and mechanical stability of the cell wall matrix. This work indicates that specific genetically encoded physical and mechanical properties of the cell wall and leaf tissues that depend on cold hardening are factors that can determine plant resistance against fungal diseases.     

Forensic pathology of canadian bread wheat: the case of tan spot

ABSTRACT Pyrenophora tritici-repentis causes necrosis and chlorosis in its wheat host. Susceptibility to races 2 (necrosis) and 5 (chlorosis) of the pathogen is known to be mediated by Ptr ToxA and Ptr ToxB, respectively. Sensitivity to each toxin is controlled by a single dominant and independently inherited gene. We used sensitivity to Ptr ToxA and Ptr ToxB as two genetic markers to investigate the origin and the state of tan spot susceptibility in Canadian Western Red Spring (CWRS) wheat over a period of more than a century. Sensitivity to Ptr ToxA, the toxin produced by nearly all isolates of the pathogen collected in the past 20 years in western Canada, appears to have been present in the first major cultivar, Red Fife, grown massively in the late 1800s. Sensitivity then was transmitted unknowingly into Canadian wheat lines through extensive use of backcrossing to maintain the Marquis-Thatcher breadmaking quality. Sensitivity to Ptr ToxA, which nearly disappeared from cultivars grown in western Canada in the 1950s, was reintroduced in the 1960s and unintentionally bred into many of the present-day cultivars. Sensitivity to Ptr ToxB, a toxin rarely found in isolates from western Canada, appeared with the release of Thatcher in 1934 and was transferred to many cultivars through backcross programs. In spite of large areas planted to Ptr ToxAand Ptr ToxB-sensitive cultivars over decades, tan spot epidemics remained sporadic until the 1970s. The results of this study raise the problem of the narrowing genetic base of CWRS wheat lines and the potential for unanticipated threats from plant pathogens. The intercrossing of genetically diverse material in one Canadian wheat breeding program resulted in the release of several modern cultivars with resistance to tan spot. The absence of wild-type Ptr ToxB-producing isolates in western Canada remains unexplained, given that sensitivity to Ptr ToxB was present continuously in western Canadian cultivars grown on vast areas for more than 70 years.     

The Identification of Two New Races of Pyrenophora tritici-repentis from the Host Center of Diversity Confirms a One-to-One Relationship in Tan Spot of Wheat

ABSTRACT Pyrenophora tritici-repentis, causal agent of tan spot, induces necrosis and chlorosis in its wheat host. The tan spot system conforms to the toxin model and three host-specific toxins have been identified (Ptr ToxA, Ptr ToxB, and putative Ptr ToxC). Processing of a collection of isolates, obtained in the Fertile Crescent and Caucasus regions, yielded two new virulence patterns. Isolate Az35-5 combined the virulences of races 2 and 5 and was classified in the new race 7. Isolates TS93-71B and TS93-71F had a virulence pattern that combined those of races 2, 3, and 5 and were grouped in the new race 8. Southern analysis revealed that all three isolates possessed copies of the ToxA and ToxB genes, the first time the genes were found in a common background. The production of Ptr ToxA and Ptr ToxB by the isolates was confirmed by western blotting. Virulence patterns suggested that TS93-71B and TS93-71F may also produce Ptr ToxC, even though it was not present at detectable levels in culture filtrates. The identification of races 7 and 8 complete the theoretical maximum number of races that can be differentiated by three loci in the host (2(3) = 8), assuming a one-to-one relationship. It appears that the wheat/P. tritici-repentis system is a mirror image of the classical gene-for-gene relationship.     

Components of quantitative resistance to leaf rust in wheat cultivars: diversity,variability and specificity

The aim of this study was to investigate the potential diversity and pathogen‐specificity of sources of quantitative resistance to leaf rust caused by Puccinia triticina in French wheat germplasm. From a set of 86 genotypes displaying a range of quantitative resistance levels during field epidemics, eight wheat genotypes were selected and challenged in a greenhouse with three isolates of the pathogen, belonging to different pathotypes. Five components of resistance were assessed: infection efficiency, for which an original methodology was developed, latent period, lesion size, spore production per lesion, and spore production per unit of sporulating tissue. High diversity and variability for all these components were expressed in the host × pathotype combinations investigated; isolate‐specificity was found for all the components. The host genotypes displayed various resistance profiles, based on both the components affected and the isolate‐specificity of the interaction. Their usefulness as sources of quantitative resistance was assessed: line LD7 probably combines diversified mechanisms of resistance, being highly resistant for all the components, but displaying isolate‐specificity for all the components; cv. Apache did not show isolate specificity for any of the components, which could be related to the durability of its quantitative resistance in the field over more than 11 years.     

Infection processes and soft wheat response to root rot and crown rot caused by Fusarium culmorum

An isolate of the fungus Fusarium culmorum constitutively expressing green fluorescent protein was used to investigate the infection process and host response of primary seedling roots and stem base leaf sheaths of soft wheat cv. Genio. Disease progress was assessed macroscopically by visual symptoms, microscopically by confocal laser scanning microscopy (CLSM) and via gene expression analysis of fungal and wheat genes by real‐time quantitative RT‐PCR. In the roots, CLSM investigations revealed an initial intercellular and subsequent intracellular colonization by fungal hyphae. The fungus invaded the rhizodermal layer and cortex but was not seen to colonize the stele. The fungus consistently expressed TRI5 (24, 48 and 96 h post‐inoculation), indicating that trichothecenes were being synthesized throughout this phase of infection and colonization. The expression of the six host defence‐associated genes (Wheatwin 1‐2, PR1, Chitinase, PAL, WIR1 and LOX) increased early in infection and decreased during later stages. In the stem base, CLSM observations revealed the fungus sequentially penetrating though the first, second and third basal leaf sheaths. Expression of TRI5 was initiated early in the infection of each leaf sheath. The expression of the host defence‐associated genes varied over time and across leaf sheaths, and all were also expressed in leaf sheaths which had not yet been in contact with the fungus. Expression of LOX and WIR1 were particularly enhanced in the third leaf sheath.     

Wax matters: absence of very‐long‐chain aldehydes from the leaf cuticular wax of the glossy11 mutant of maize compromises the prepenetration processes of Blumeria graminis

Conidial germination and differentiation, the so‐called prepenetration processes, of the barley powdery mildew fungus (Blumeria graminis f.sp. hordei) are triggered in vitro by very‐long‐chain aldehydes, minor constituents of barley leaf wax. However, until now it has not been demonstrated that these cuticle‐derived molecules also play a significant role in the initiation and promotion of the fungal prepenetration processes in vivo, on the surface of a living plant leaf. In the maize (Zea mays) wax mutant glossy11, which is completely devoid of cuticular very‐long‐chain aldehydes, germination and appressorial differentiation of B. graminis were strongly impeded. Spraying the mutant leaf surface with aldehyde‐containing wild‐type wax or pure n‐hexacosanal (C₂₆‐aldehyde) fully restored fungal prepenetration, whereas maize wild‐type leaf surfaces coated with n‐docosanoic acid exhibited reduced conidial germination rates of 23%, and only 5% of the conidia differentiated infection structures. In vitro studies were performed to further corroborate the extensive prevention of fungal germination and differentiation in response to artificial surfaces coated with aldehyde‐deficient maize wax. Because of its phenotype affecting the B. graminis prepenetration processes, the glossy11 mutation of maize may become a valuable molecular target and genetic tool that could provide a means of developing basal powdery mildew resistance in the globally important crops wheat and barley.     

Assessment of the pest status of Pratylenchus curvicauda and ultrastructural changes in roots of infected wheat and barley

Pratylenchus curvicauda, which was first described in metropolitan Perth in 1991, was recently identified in grain-growing areas in Western Australia. The biology of this root-lesion nematode, and especially its pest status, is unknown. We investigated its life cycle and interaction with host plants, because such information is essential for its management. The life cycle took 45 days to complete in a wheat cultivar maintained at 23°C. Over 10 weeks, the nematode multiplied in 26 of 61 genotypes; these host plants were all cereals and included widely grown cultivars of wheat and barley. Eighteen other cereal genotypes and 13 cultivars including canola, chickpea, ryegrass, lupin, soybean, and tomato, sustained the nematodes to different degrees without multiplication. Four cover crops were not suitable hosts. The patterns of attraction of the nematodes and penetration into roots of the host and tolerant plants were similar. The nonhosts attracted fewer nematodes, none of which penetrated the roots. Browning of infected roots was atypical—it occurred late in some roots, 55 days after inoculation, and in the presence of a fungus. The nematodes were confined to, and fed from, cortical cells. The ultrastructure of infected wheat and barley cells showed typical signs of damage caused by Pratylenchus spp. and included cell disorganization and lack of membrane integrity, disintegration of cytoplasm, hypertrophy of some nuclei, and deposition of tannin-like granules. This detailed characterization of P. curvicauda–host interaction indicates the nematode is likely to be a pest of major crops, and attention should be given to its management.     

Testing and modelling the potential of three diploid plants in Poaceae as a new pathosystem to investigate the interactions between cereal hosts and cereal cyst nematode (Heterodera avenae)

Cereal cyst nematode (CCN), Heterodera avenae, is one of the most important pathogens of wheat worldwide, and causes significant yield losses. Research on CCN–wheat interactions is hampered by the lack of an effective model pathosystem. This study investigated the potential of the model cereal Brachypodium distachyon (Bd21‐3) and diploid wheat 2A (G1812) and 2D (AL8/78) as model hosts for CCN. Nematode infection analysis showed that although some CCN penetrated Bd21‐3 roots, these nematodes failed to develop to the later developmental stages or form cysts, indicating B. distachyon is not a host for CCN. A strong burst of reactive oxygen species (ROS) within Bd21‐3 roots infected with CCN was induced 3 days after infection and the expression of seven ROS‐producing genes was significantly increased. In contrast, CCN completed its life cycle in both diploid wheat 2A and 2D, and formed normal syncytia in these hosts. Although CCN developmental processes within both diploid wheat 2A and 2D were very similar to those in the susceptible control, the number of cysts formed on diploid wheat 2D was less than those formed on diploid wheat 2A and the susceptible control, indicating that diploid wheat 2A was a more suitable host for CCN than 2D. This is the first report of a potential new pathosystem for CCN–host interactions using diploid wheat.     

Effect of wheat spike infection timing on fusarium head blight development and mycotoxin accumulation

Fusarium head blight in wheat spikes is associated with production of mycotoxins by the fungi. Although flowering is recognized as the most favourable host stage for infection, a better understanding of infection timing on disease development and toxin accumulation is needed. This study monitored the development of eight characterized isolates of F. graminearum, F. culmorum and F. poae in a greenhouse experiment. The fungi were inoculated on winter wheat spikes before or at anther extrusion, or at 8, 18 and 28 days later. Disease levels were estimated by the AUDPC and thousand‐kernel weight (TKW). The fungal biomass (estimated by qPCR) and toxin concentration (deoxynivalenol and nivalenol, estimated by UPLC‐UV‐MS/MS) were measured in each inoculated spike, providing a robust estimation of these variables and allowing correlations based on single‐individual measurements to be established. The toxin content correlated well with fungal biomass in kernels, independently of inoculation date. The AUDPC was correlated with fungal DNA, but not for early and late infection dates. The highest disease and toxin levels were for inoculations around anthesis, but early or late infections led to detectable levels of fungus and toxin for the most aggressive isolates. Fungal development appeared higher in kernels than in the chaff for inoculations at anthesis, but the opposite was found for later inoculations. These results show that anthesis is the most susceptible stage for FHB, but also clearly shows that early and late infections can produce significant disease development and toxin accumulation with symptoms difficult to estimate visually.     

Host‐induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens.     

The importance of non‐penetrated papillae formation in the resistance response of triticale to powdery mildew (Blumeria graminis)

Triticale is the intergeneric hybrid between wheat and rye. With the expansion of the triticale growing area, powdery mildew has emerged and become a significant disease on this new host. Recent research demonstrated that this ‘new’ powdery mildew on triticale has emerged through a host range expansion of powdery mildew of wheat. Moreover, isolates sampled from triticale still infect their previous host, wheat, but isolates sampled from wheat hardly infect triticale. Race‐specific and adult‐plant resistance have been identified in triticale cultivars. The main objective of this study was to characterize the cellular basis of powdery mildew resistance in triticale. Commonalities with resistance responses in other cereals such as wheat, barley and oat are discussed. A detailed comparative histological study of various resistance responses during cross‐inoculation of either virulent or avirulent wheat and triticale isolates on both hosts was carried out. The present data provide evidence that for incompatible interactions, the formation of non‐penetrated papillae is the predominant resistance response, while the hypersensitive response (HR) acts as a second line of defence, to cut the fungus off from nutrients, if penetration resistance fails. It is not clear yet what causes the slower growth and reduced colony size of triticale isolates when inoculated on wheat. Possibly, post‐penetration resistance mechanisms, other than HR, are switched on during these (semi‐) compatible interactions. Molecular studies on gene expression and gene function of defence‐related genes might reveal further insights into the genetic basis of these resistance responses.     

A Combination of Phenotypic and Genotypic Characterization Strengthens Pyrenophora tritici-repentis Race Identification

ABSTRACT Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces multiple host-selective toxins (HSTs), including Ptr ToxA, Ptr ToxB, and Ptr ToxC. The specific complement of HSTs produced by a particular isolate determines its host cultivar specificity. Each unique specificity profile, represented by the differential induction of necrosis or chlorosis on a standard set of wheat differentials, defines a unique race. Eight races of P. tritici-repentis have been formally published, although additional races are under investigation. Although visual assessment of disease phenotype is often used in race designation of P. tritici-repentis, our results suggest that it has the potential to be misleading. Inoculation of the P. tritici-repentis isolates SO3 and PT82 on the current wheat differential set indicated classification as race 2 and race 8, respectively; however, genetic characterization revealed that these isolates do not possess the associated HSTs expected for these race assignments. Despite sharing disease phenotypes similar to known races, SO3 and PT82 were genotypically distinct from these previously characterized races of P. tritici-repentis. To ensure detection of the breadth of physiological variation among the isolates of P. tritici-repentis, our results indicate that race classification, where possible, should include both phenotypic and genotypic analyses and eventual expansion of the differential set.     

Virulence assessment of Australian Pyrenophora tritici-repentis isolates

The virulence of 57 Australian isolates of Pyrenophora tritici-repentis (Ptr), a necrotrophic fungal pathogen responsible for the major wheat disease tan spot, was assessed through plant infection assays. Isolates collected from the northern, southern, and western wheat-cropping regions of Australia were evaluated against 16 Australian bread wheat cultivars under controlled growth conditions. Following infection, the wheat panel displayed varying disease symptoms ranging from tiny necrotic specks to spreading chlorotic and necrotic lesions. Analysis of variance indicated that the wheat cultivar exhibited a greater effect on the disease response, explaining 62.7% of the variation, in comparison to the isolate (10.4%). The interaction between the cultivar and the isolate was statistically significant and was attributed to 9.8% of the total variation. All Ptr isolates examined were able to cause disease, but did not display a clear distinction in virulence on the wheat panel investigated, instead showing subtle differences in aggressiveness. Based on the disease responses, there was no obvious pattern between isolate aggressiveness and cropping region. Some cultivars, such as Hydra, exhibited an effective level of resistance in relation to the panel of isolates tested. All 57 Ptr isolates were found to possess the ToxA effector gene and lack the ToxB effector gene. The gene expression level of ToxA was up-regulated at 3 days postinfection in both ToxA-sensitive and -insensitive cultivars, independent of ToxA–Tsn1 recognition.     

Molecular characterization,comparison of screening methods,and evaluation of cross‐pathogenicity of black rot (Xanthomonas campestris pv. campestris) strains from cabbage,choy sum,leafy mustard and pak choi from Taiwan

Choy sum (Brassica rapa var. parachinensis), leafy mustard (Brassica juncea) and pak choi (B. rapa var. chinensis) are highly nutritious components of diets in Taiwan and other Asian countries, and bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) is a major biotic constraint in these crops. As very little was known about the Xcc strains from these crops in these regions, including their cross‐pathogenicity and aggressiveness on different hosts, Xcc strains were obtained from cabbage (Brassica oleracea var. capitata), choy sum, leafy mustard and pak choi crops in Taiwan. Two previously published PCR‐based assays reliably distinguished the Xcc strains from other Xanthomonas species and subspecies. Phylogenetic analysis based on repetitive sequence‐based PCR assays placed the Xcc strains in a clade distinct from other Xanthomonas species, and also showed host specificity. Although all of the Xcc strains from the different host species were pathogenic on all five Brassica test species in both a detached leaf assay and an intact plant assay, in the intact plant assay they showed differences in virulence or aggression on the different test hosts. The Xcc strains from leafy mustard and pak choi were consistently highly aggressive on all the test host genotypes, but the strains from choy sum and cabbage were less aggressive on leafy mustard and choy sum. The intact plant assay proved more discriminating and reliable than the detached leaf assay for comparing the aggressiveness of Xcc strains on different host genotypes, and so, with the new Xcc strains isolated in this study, will be useful for screening leafy brassica germplasm accessions for resistance to black rot.