Comparative analysis reveals changes in transcriptomes of sugarcane upon infection by Leifsoniaxyli subsp. xyli

Ratoon stunting disease (RSD) caused by bacterium Leifsoniaxyli subsp. xyli (Lxx) is a devastating disease of sugarcane over a large part of the world. Genetic improvement for RSD‐resistant varieties is considered the most effective method to control the disease. However, genetic improvement of sugarcane is hindered by the limited information about the molecular mechanisms underlying Lxx pathogenicity and defence responses in sugarcane. In this study, genome‐wide gene expression profiling was used to compare RSD‐resistant (CP72‐2086) and RSD‐susceptible (GT11) genotypes at different infection time points in order to identify the candidate regulators for RSD resistance. A total of 14,494 differentially expressed genes (DEGs) were identified, indicating that dramatic changes had occurred in gene expression upon Lxx infection, especially in the susceptible genotype. Enrichment analysis showed that a large number of genes related to plant hormone signal transduction, phenylalanine metabolism, phenylpropanoid biosynthesis and starch and sucrose metabolism was responsible for sugarcane response to Lxx infection. Plant hormone signalling pathway genes were significantly differentially expressed at the early infection stage between the two genotypes. The resistant genotype chose the jasmonic acid‐ and ethylene‐dependent host‐defence pathways to resist Lxx infection, whereas the susceptible genotype preferred the salicylic acid‐dependent host‐defence pathways. These findings help unravel the molecular mechanisms of sugarcane plant–Lxx interactions and may pave the way for sugarcane breeding for disease resistance.     

Physiological Basis for Differences in Resistance to Microdochium nivale (Fr.) Samuels and Hallett in Two Androgenic Genotypes of Festulolium Derived from Tetraploid F1 Hybrids of Festuca pratensis × Lolium multiflorum (Festulolium)

The aim of this study was to investigate the physiological basis for differences in resistance to pink snow mould (Microdochium nivale) in two androgenic genotypes of Festulolium (Festuca pratensis × Lolium multiflorum) which differed in terms of their resistance to M. nivale. Genotype 716 was more resistant than genotype 729. The study consisted of two experiments. The aim of the first experiment was to estimate the ability of the plants to survive winter conditions. The aim of the second experiment was to find physiological markers of resistance to snow mould. Festulolium plants were infected with M. nivale mycelium after pre‐hardening and hardening. After 2 weeks in the dark at 2°C, there was a sharp increase in the phenolic content in both genotypes. The increase was greater in the more resistant genotype 716 than in genotype 729. Phenolics therefore may play a very important role in overwintering in grasses, similar to carbohydrates. Based on the differences between the two genotypes, potential indicators of resistance to M. nivale in Festulolium include increased soluble carbohydrate content, increased phenolic content, increased hydrogen peroxide accumulation, decreased catalase activity, increased abscisic acid content and reduced heat emission.     

The Effect of Chitosan and Bion on Resistance to Pink Snow Mould in Perennial Ryegrass and Winter Wheat

The effects of chitosan on resistance to pink snow mould (Microdochium nivale) were studied in young winter wheat (Triticum aestivum L.) and perennial ryegrass (Lolium perenne L.) under controlled environmental conditions. In perennial ryegrass, the putative defence activator Bion was also tested. Resistance was measured as regrowth of plants after inoculation with M. nivale and incubation in darkness at 2°C. In winter wheat, pre‐treatment with chitosan at 1000 μg per plant increased resistance to subsequent infection by M. nivale, but this effect was less significant in a replicate experiment. Chitosan‐treated winter wheat plants expressed the gene for the pathogenesis‐related protein chitinase at higher levels than non‐treated plants. Chitinase gene expression was also stimulated by M. nivale infection in winter wheat. Perennial ryegrass pre‐treated with Bion or chitosan and inoculated with M. nivale did not display better regrowth after incubation than non‐treated, inoculated plants. Rather, regrowth was reduced in some of the Bion‐treated plants after incubation. We speculate that the cost or the mechanism of induced resistance makes Bion non‐effective in plants that are not actively growing. Bion at concentrations of 10, 100 and 1000 μg active ingredient per ml, and the highest concentration of chitosan used (2000 μg per ml) reduced in vitro growth of the pathogen, suggesting that both defence activators possess antifungal activity.     

<Emphasis Type="Italic">Microdochium nivale</Emphasis> (Fr., Samuels &; Hallett): cytological analysis of the infection process in triticale (×<Emphasis Type="Italic">Triticosecale</Emphasis> Wittm.)

According to regular reports, one of the most serious diseases of winter cereal and grass varieties in moderate and cold climatic areas is pink snow mould caused by Microdochium nivale. Currently, the resistance of the economically important cereal species as triticale is not satisfactory. Moreover, there is no efficient strategy of protection against this pathogen and the understanding of plant resistance mechanisms is rather poor. Presented paper for the first time shows the cytological analysis of M. nivale infection in model triticale varieties by the use of fluorescent and light microscopy in combination with fluorescent dyes and hydrogen peroxide staining. Both, the infection level and the dynamic of the process varied for tested genotypes confirming the field and laboratory data of their different resistance to this pathogen. Moreover, our analysis showed that in both cultivars cold-hardening of seedlings delayed the mycelium growth. In both cultivars, hyphal walls and fungal penetration sites were visualized in crowns, leaf sheaths and leaves of hardened and non-hardened inoculated seedlings. For the first time the presence of the haustoria produced by M. nivale was confirmed in those tissues. Single infection hyphae usually penetrated into the host tissues via stomatal apparatuses were accompanied by the efflux of hydrogen peroxide. The data show a great potential of fluorescence techniques in studying the host plant–pathogen interactions providing a better insight into plant defence reactions that may allow elaboration of the efficient breeding strategies aimed at increasing resistance to this pathogenic fungus.     

Elevated CO2 changes the interactions between nematode and tomato genotypes differing in the JA pathway

Interactions between the root‐knot nematode Meloidogyne incognita and three isogenic tomato (Lycopersicon esculentum) genotypes were examined when plants were grown under ambient (370 ppm) and elevated (750 ppm) CO₂. We tested the hypothesis that, defence‐recessive genotypes tend to allocate ‘extra’ carbon (relative to nitrogen) to growth under elevated CO₂, whereas defence‐dominated genotypes allocate extra carbon to defence, and thereby increases the defence against nematodes. For all three genotypes, elevated CO₂ increased height, biomass, and root and leaf total non‐structural carbohydrates (TNC):N ratio, and decreased amino acids and proteins in leaves. The activity of anti‐oxidant enzymes (superoxide dismutase and catalase) was enhanced by nematode infection in defence‐recessive genotypes. Furthermore, elevated CO₂ and nematode infection did not qualitatively change the volatile organic compounds (VOC) emitted from plants. Elevated CO₂ increased the VOC emission rate only for defence‐dominated genotypes that were not infected with nematodes. Elevated CO₂ increased the number of nematode‐induced galls on defence‐dominated genotypes but not on wild‐types or defence‐recessive genotypes roots. Our results suggest that CO₂ enrichment may not only increase plant C : N ratio but can disrupt the allocation of plant resources between growth and defence in some genetically modified plants and thereby reduce their resistance to nematodes.     

Cold-hardening of winter triticale (<Emphasis Type="Italic">x Triticosecale</Emphasis> Wittm.) results in increased resistance to pink snow mould <Emphasis Type="Italic">Microdochium nivale</Emphasis> (Fr., Samuels &; Hallett) and genotype-dependent chlorophyll fluorescence modulations

The resistance of triticale (x Triticosecale Wittm.) to infection of snow mould Microdochium nivale (Fr., Samuels & Hallett) was examined under different temperature pre-treatment regimes. The results of laboratory “cold chamber” resistance tests correlated with the breeders’ report from field experiments. Studied genotypes differed substantially in their resistance to infection. Two cultivars: ‘Magnat’ (susceptible) and ‘Hewo’ (relatively resistant) were further studied as a plant model to test the role of pre-hardening and cold-hardening induction of resistance expression. Both model cultivars were susceptible to M. nivale infection without cold pre-treatment and gained genotype-depended level of resistance after 4 weeks treatment at 4°C, moreover the resistance grew gradually. Simultaneously to the resistance tests, the measurements of chlorophyll fluorescence parameters were taken. The results showed that higher vitality index Rfd of cold-hardened triticale seedlings correlated with increased pink snow mould resistance while differences in other parameters of fluorescence were not distinctly significant. Establishment of Rfd in 4 weeks hardened triticale seedlings could be used for a large scale screening of breeding material in order to select potentially resistant genotypes. Such analyses have not been reported for triticale before.     

Infection with virulent and avirulent P. syringae strains differentially affects photosynthesis and sink metabolism in Arabidopsis leaves

Infection of plants with pathogens leads not only to the induction of defence reactions but also to changes in carbohydrate metabolism. In this study, the effects of infection by a virulent and an avirulent strain of P. syringae on spatio-temporal changes in photosynthesis were compared using chlorophyll fluorescence imaging. The maximum PSII quantum yield, effective PSII quantum yield and nonphotochemical quenching were decreased in Arabidopsis leaves infected with either strain. At the same time, the quantum yield of nonregulated energy dissipation was increased. These changes could be detected by chlorophyll fluorescence imaging before symptoms were visible by eye. The effects were restricted to the vicinity of the infection site and did not spread to uninfected areas of the leaf. Qualitatively similar changes in photosynthetic parameters were observed in both interactions. Major differences between the responses to both strains were evident in the onset and time course of changes. A decrease in photosynthesis was detectable already at 3 h only after challenge with the avirulent strain while after 48 h the rate of photosynthesis was lower with the virulent strain. In contrast to photosynthesis, the regulation of marker genes for source/sink relations and the activities of invertase isoenzymes showed qualitative differences between both interactions. Inoculation of the virulent but not the avirulent strain resulted in downregulation of photosynthetic genes and upregulation of vacuolar invertases. The activity of vacuolar invertases transiently increased upon infection with the virulent strain but decreased with the avirulent strain while extracellular invertase activity was downregulated in both interactions.     

Do virus‐resistant plants pose a threat to non‐target ecosystems? II. Risk assessment of an Australian pathosystem using multi‐scale field experiments

It has been widely argued that the acquisition of novel disease resistance genes by wild host populations following the release of novel pathogen‐resistant plants into agricultural systems could pose a significant threat to non‐target plant communities. However, predicting the magnitude of ecological release in wild plant populations following the removal of disease remains a major challenge. In this paper we report on the second phase of a tiered risk assessment designed to investigate the role of disease on host growth, survival, fecundity and fitness in a model pathosystem (the pasture species Trifolium repens infected with Clover yellow vein virus, ClYVV) and to assess the level of risk posed to at‐risk native plant communities in southeast Australia by newly developed genetically modified and conventionally bred virus‐resistant T. repens genotypes. Multi‐year field experiments conducted in woodland and grassland environments using host‐pathogen arrays derived from 14 ClYVV isolates and 21 T. repens genotypes indicate that viral infection reduces fecundity, growth and survival of wild T. repens plants but that the severity of these effects depends on host tolerance to infection, isolate aggressiveness and specific spatial and temporal environmental conditions. Demographic modelling showed that by reducing host survival and growth, ClYVV also limits the intrinsic population growth rate and niche size of wild T. repens populations. Given the significant fitness cost associated with viral infection we conclude that virus‐resistant T. repens genotypes may pose a threat to some high conservation‐value non‐target ecosystems in SE Australia. We also argue that long‐term, multi‐tiered experiments conducted in a range of controlled and non‐controlled environments are necessary to detect and accurately quantify risks associated with the release of disease‐resistant plants in general.     

Do virus‐resistant plants pose a threat to non‐target ecosystems? I. Evidence from an Australian pathosystem based on glasshouse challenge experiments

One key environmental risk associated with the release of novel disease‐resistant plants is the potential for non‐target host populations to acquire resistance genes and undergo enemy release, leading to damage to associated native plant populations in high conservation‐value ecosystems. Unfortunately, the dynamics of most natural pathosystems are poorly understood, and risk assessment of disease‐resistant plants remains a challenge. Here we describe the first stage of a multi‐tiered risk assessment strategy aimed at quantifying potential ecological release in a model pathosystem (the weedy pasture species Trifolium repens infected with Clover yellow vein virus; ClYVV) in order to assess the level of risk posed by genetically modified and conventionally bred disease‐resistant host genotypes to non‐target plant communities in south‐eastern Australia. Glasshouse inoculation and growth experiments using 14 ClYVV isolates and 20 wild T. repens lines collected from high conservation‐value montane grassland and woodland communities show that viral infection reduces the survival and growth of host plants by on average 10–50%. However, T. repens lines exhibited variable levels of resistance and tolerance to virus infection and ClYVV isolates differed in infectivity and aggressiveness, with grassland isolates having a greater pathogenic effect on associated host plants than woodland isolates. We conclude that ClYVV potentially plays an important role in limiting the size of T. repens populations in some at‐risk non‐target ecosystems and that second‐tier field experiments are required to adequately quantify the risk associated with the commercial release of V‐R T. repens genotypes in Australia.     

Identification of additional sources of resistance to Puccinia striiformis f. sp. hordei (PSH) in a collection of barley genotypes adapted to the high input condition

A total of 336 barley genotypes consisting of released cultivars, advanced lines, differentials and local landraces from the ICARDA barley breeding programme were screened for seedling and adult‐plant resistances to barley stripe rust pathogen (Puccinia striiformis f. sp. hordei [PSH]). Seedling resistance tests were undertaken at Shimla, India by inoculating 336 barley genotypes with five prevalent PSH races [Q (5S0), 24 (0S0‐1), 57 (0S0), M (1S0) and G (4S0)] in India. Barley genotypes were also evaluated at the adult‐plant stage for stripe rust resistance at Durgapura (Rajasthan, India) in 2013 and 2014, and at Karnal (Haryana, India) in 2014 under artificial PSH infection in fields, using a mixture of the five races. Twelve barley genotypes (ARAMIR/COSSACK, Astrix, C8806, C9430, CLE 202, Gold, Gull, Isaria, Lechtaler, Piroline, Stirling, and Trumpf) were resistant to all five PSH races at the seedling and adult‐plant stages. Two of these genotypes, Astrix and Trumpf, were part of international differentials and reveal that five races were avirulent to genes Rps4 (yr4), rpsAst, rpsTr1 and rpsTr2. These genes were highly effective against PSH races prevalent in India. The virulence/avirulence formula reported in this study helped to determine the effectiveness of PSH resistance genes against Indian races. Forty‐five genotypes showed adult‐stage plant resistance (APR) in the field. The identified PSH resistant genotypes may possess novel resistance genes and might serve as potential donors of PSH resistance at seedling and APR in the future. Further research is needed to determine the nature of resistance genes through allelic studies and mapping of these genes.     

Resistance and biomass in Arabidopsis: a new model for Salicylic Acid perception

Salicylic acid (SA) is an essential hormone for plant defence and development. SA perception is usually measured by counting the number of pathogens that grow in planta upon an exogenous application of the hormone. A biological SA perception model based on plant fresh weight reduction caused by disease resistance in Arabidopsis thaliana is proposed. This effect is more noticeable when a chemical analogue of SA is used, like Benzothiadiazole (BTH). By spraying BTH several times, a substantial difference in plant biomass is observed when compared with the mock treatment. Such difference is dose‐dependent and does not require pathogen inoculation. The model is robust and allows for the comparison of different Arabidopsis ecotypes, recombinant inbreed lines, and mutants. Our results show that two mutants, non‐expresser of pathogenesis‐related genes 1 (npr1) and auxin resistant 3 (axr3), fail to lose biomass when BTH is applied to them. Further experiments show that axr3 responds to SA and BTH in terms of defence induction. NPR1‐related genotypes also confirm the pivotal role of NPR1 in SA perception, and suggest an active program of depletion of resources in the infected tissues.     

Nitrogen modulation of Medicago truncatula resistance to Aphanomyces euteiches depends on plant genotype

Nitrogen (N) availability can impact plant resistance to pathogens by the regulation of plant immunity. To better understand the links between N nutrition and plant defence, we analysed the impact of N availability on Medicago truncatula resistance to the root pathogen Aphanomyces euteiches. This oomycete is considered to be the most limiting factor for legume production. Ten plant genotypes were tested in vitro for their resistance to A. euteiches in either complete or nitrate‐deficient medium. N deficiency led to enhanced or reduced susceptibility depending on the plant genotype. Focusing on four genotypes displaying contrasting responses, we determined the impact of N deficiency on plant growth and shoot N concentration, and performed expression analyses on N‐ and defence‐related genes, as well as the quantification of soluble phenolics and different amino acids in roots. Our analyses suggest that N modulation of plant resistance is not linked to plant response to N deprivation or to mechanisms previously identified to be involved in plant resistance. Furthermore, our studies highlight a role of glutamine in mediating the susceptibility to A. euteiches in M. truncatula.