Sulphate and sulphurous acid alter the relative susceptibility of wheat to Phaeosphaeria nodorum and Mycosphaerella graminicola

The relative abundances of DNA of Mycosphaerella graminicola and Phaeosphaeria nodorum in archived wheat samples are closely correlated with UK anthropogenic emissions of oxidized sulphur over the last 160 years. To test whether this could be a causal relationship, possible modes of action of sulphur on the two fungi were examined. Mycelial growth of the two fungi in solutions of sulphurous acid was similar. Sulphurous acid at pH 4 reduced percentage germination of P. nodorum conidia more strongly than M. graminicola conidia. In spray inoculations of wheat cv. Squarehead’s Master, Cappelle Desprez and Riband with water or sulphurous acid (pH 4), the ratio of leaves infected by P. nodorum to leaves infected by M. graminicola was increased by factors of 2.5, 2.1 and 0.6, respectively at pH 4. The same three cultivars of wheat were grown in sand and vermiculite and fertilized with nutrient solution containing 2.5 or 0.5 mM sulphate. Both pathogens infected less frequently at 2.5 mM sulphate, by a factor of about 2. The severity of infection by M. graminicola was reduced on all three cultivars by a factor of about 4-5 at 2.5mM sulphate, but severity of P. nodorum was reduced only by a factor of about 2. Both elevated free sulphate concentrations in soil and sulphite in rainwater could therefore increase the prevalence of P. nodorum relative to M. graminicola, which is consistent with the historical changes in abundance

Evidence that nematodes may vector the soft rot-causing enterobacterial phytopathogens

Bacterial soft rot is a globally significant plant disease that causes major losses in the production of many popular crops, such as potato. Little is known about the dispersal and ecology of soft-rot enterobacteria, and few animals have been identified as vectors for these pathogens. This study investigates whether soil-living and bacterial-feeding nematodes could act as vectors for the dispersal of soft-rot enterobacteria to plants. Soft-rot enterobacteria associated with nematodes were quantified and visualized through bacterial enumeration, GFP-tagging, and confocal and electron scanning microscopy. Soft-rot enterobacteria were able to withstand nematode grazing, colonize the gut of Caenorhabditis elegans and subsequently disperse to plant material while remaining virulent. Two nematode species were also isolated from a rotten potato sample obtained from a potato storage facility in Finland. Furthermore, one of these isolates (Pristionchus sp. FIN-1) was shown to be able to disperse soft-rot enterobacteria to plant material. The interaction of nematodes and soft-rot enterobacteria seems to be more mutualistic rather than pathogenic, but more research is needed to explain how soft-rot enterobacteria remain viable inside nematodes.

Alterations in predicted regulatory and coding regions of the sterol 14α-demethylase gene (CYP51) confer decreased azole sensitivity in the oilseed rape pathogen Pyrenopeziza brassicae

The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops is increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivities of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to sub-inhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting PbCYP51 evolution is on-going and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.

Botrytis species: relentless necrotrophic thugs or endophytes gone rogue?

Plant pathology has a long-standing tradition of classifying microbes as pathogens, endophytes or saprophytes. Lifestyles of pathogens are categorized as biotrophic, necrotrophic or hemibiotrophic. Botrytis species are considered by many to be archetypal examples of necrotrophic fungi, with B. cinerea being the most extensively studied species because of its broad host range and economic impact. In this review, we discuss recent work which illustrates that B. cinerea is capable of colonizing plants internally, presumably as an endophyte, without causing any disease or stress symptoms. The extent of the facultative endophytic behaviour of B. cinerea and its relevance in the ecology and disease epidemiology may be vastly underestimated. Moreover, we discuss the recent discovery of a novel Botrytis species, B. deweyae, which normally grows as an endophyte in ornamental daylilies (Hemerocallis), but displays facultative pathogenic behaviour, and is increasingly causing economic damage. We propose that the emergence of endophytes ‘gone rogue’ as novel diseases may be related to increased inbreeding of hybrid lines and reduced genetic diversity. These observations lead us to argue that the sometimes inflexible classification of pathogenic microbes by their lifestyles requires serious reconsideration. There is much more variety to the interactions of Botrytis with its hosts than the eye (or the plant pathologist) can see, and this may be true for other microbes interacting with plants.

The population dynamics of disease on short and long time-scales

Observational evidence is scarce concerning the distribution of plant pathogen population sizes or densities as a function of time-scale or spatial scale. For wild pathosystems we can only get indirect evidence from evolutionary patterns and the consequences of biological invasions.We have little or no evidence bearing on extermination of hosts by pathogens, or successful escape of a host from a pathogen. Evidence over the last couple of centuries from crops suggest that the abundance of particular pathogens in the spectrum affecting a given host can vary hugely on decadal timescales. However, this may be an artefact of domestication and intensive cultivation. Host-pathogen dynamics can be formulated mathematically fairly easily–for example as SIR-type differential equation or difference equation models, and this has been the (successful) focus of recent work in crops. “Long-term” is then discussed in terms of the time taken to relax from a perturbation to the asymptotic state. However, both host and pathogen dynamics are driven by environmental factors as well as their mutual interactions, and both host and pathogen co-evolve, and evolve in response to external factors. We have virtually no information about the importance and natural role of higher trophic levels (hyperpathogens) and competitors, but they could also induce long-scale fluctuations in the abundance of pathogens on particular hosts. In wild pathosystems the host distribution cannot be modelled as either a uniform density or even a uniform distribution of fields (which could then be treated as individuals). Patterns of short term density-dependence and the detail of host distribution are therefore critical to long-term dynamics. Host density distributions are not usually scale-free, but are rarely uniform or clearly structured on a single scale. In a (multiply structured) metapopulation with coevolution and external disturbances it could well be the case that the time required to attain equilibrium (if it exists) based on conditions stable over a specified time-scale is longer than that time-scale. Alternatively, local equilibria may be reached fairly rapidly following perturbations but the meta-population equilibrium be attained very slowly. In either case, meta-stability on various time-scales is a more relevant than equilibrium concepts in explaining observed patterns.

The endophytic fungus Piriformospora indica protects wheat from Fusarium crown rot disease in simulated UK autumn conditions

The root endophytic fungus Piriformospora indica (Sebacinacea) forms mutualistic symbioses with a broad range of host plants, increasing their biomass production and resistance to fungal pathogens. We evaluated the effect of P. indica on Fusarium crown rot disease of wheat, under in vitro and glasshouse conditions. Interaction of P. indica and Fusarium isolates under axenic culture conditions indicated no direct antagonistic activity of P. indica against Fusarium isolates. Seedlings of wheat were inoculated with P. indica and pathogenic Fusarium culmorum or F. graminearum and grown in sterilised soil-free medium or in a non-sterilised mix of soil and sand. Fusarium alone reduced emergence and led to visible browning and reduced root growth. Roots of seedlings in pots inoculated with both Fusarium isolates and P. indica were free of visible symptoms; seed emergence and root biomass were equivalent to the uninoculated. DNA was quantified by real-time polymerase chain reaction (qPCR). The ratio of Fusarium DNA to wheat DNA rose rapidly in the plants inoculated with Fusarium alone; isolates and species were not significantly different. P. indica inoculation reduced the ratio of Fusarium to host DNA in the root systems. The reduction increased with time. The ratio of P. indica to wheat DNA initially rose but then declined in root systems without Fusarium. With Fusarium, the ratio rose throughout the experiment. The absolute amount of Fusarium DNA in root systems increased in the absence of P. indica but was static in plants co-inoculated with P. indica.

Effect of azole fungicide mixtures, alternations and dose on azole sensitivity in the wheat pathogen Zymoseptoria tritici

The evolution of fungicide resistance in the cereal pathogen Zymoseptoria tritici, is a serious threat to the sustainability and profitability of wheat production in Europe. Application of azole fungicides has been shown to affect fitness of Z. tritici variants differentially, so it has been hypothesised that combinations of azoles could slow the evolution of resistance. This work was initiated to assess the effects of dose, mixtures and alternations of two azoles on selection for isolates with reduced sensitivity and on disease control. Naturally infected field trials were carried out at six sites across Ireland and the sensitivity of Z. tritici isolates monitored pre- and post-treatment. The azoles epoxiconazole and metconazole were applied as solo products, in alternation with each other and as a pre-formulated mixture. Full and half label doses were tested. The two azoles were partially cross-resistant, with a common azole resistance principal component accounting for 75% of the variation between isolates. Selection for isolates with reduced azole sensitivity was correlated with disease control. Decreased doses were related to decreases in sensitivity but the effect was barely significant (P = 0.1) and control was reduced. Single applications of an active ingredient (a.i.) caused smaller decreases in sensitivity than double applications. Shifts in sensitivity to the a.i. applied to a plot were greater than to the a.i. not applied, and the decrease in sensitivity was greater to the a.i. applied at the second timing. These results confirm the need to mix a.i.s with different modes of action.

Piriformospora indica reduces Fusarium head blight disease severity and mycotoxin DON contamination in wheat under UK weather conditions

Piriformospora indica (Sebacinaceae) is a cultivable root endophytic fungus. It colonises the roots of a wide range of host plants. In many settings colonisation promotes host growth, increases yield and protects the host from fungal diseases. We evaluated the effect of P. indica on Fusarium head blight (FHB) disease of winter (cv. Battalion) and spring (cv. Paragon, Mulika, Zircon, Granary, KWS Willow and KWS Kilburn) wheat and consequent contamination by the mycotoxin deoxynivalenol (DON) under UK weather conditions. Interactions of P. indica with an arbuscular mycorrhizal fungus (Funneliformis mosseae), fungicide application (Aviator Xpro) and low and high fertiliser levels were considered. P. indica application reduced FHB disease severity and incidence by 70%. It decreased mycotoxin DON concentration of winter and spring wheat samples by 70% and 80% respectively. P. indica also increased above ground biomass, 1000 grain weight and total grain weight. P. indica reduced disease severity and increased yield in both high and low fertiliser levels. The effect of P. indica was compatible with F. mosseae and foliar fungicide application. P. indica did not have any effects on plant tissue nutrients. These results suggest that P. indica might be useful in biological control of Fusarium diseases of wheat.

Clubroot (Plasmodiophora brassicae Woronin): an agricultural and biological challenge worldwide

Clubroot disease and the causal microbe Plasmodiophora brassicae offer abundant challenges to agriculturists and biological scientists. This microbe is well fitted for the environments which it inhabits. Plasmodiophora brassicae exists in soil as microscopic well protected resting spores and then grows actively and reproduces while shielded inside the roots of host plants. The pathogen is active outside the host for only short periods. Consequently, scientific studies are made challenging by the biological context of the host and pathogen and the technology required to investigate and understand that relationship. Controlling clubroot disease is a challenge for farmers, crop consultants and plant pathology practitioners because of the limited options which are available. Full symptom expression happens solely in members of the Brassicaceae family. Currently, only a few genes expressing strong resistance to P. brassicae are known and readily available. Agrochemical control is similarly limited by difficulties in molecule formulation which combines efficacy with environmental acceptability. Manipulation of husbandry encouraging improvements in soil structure, texture, nutrient composition and moisture content can reduce populations of P. brassicae. Integrating such strategies with rotation and crop management will reduce but not eliminate this disease. There are indications that forms of biological competition may be mobilised as additions to integrated control strategies. The aim of this review is to chart key themes in the development of scientific biological understanding of this host-pathogen relationship by offering signposts to grapple with clubroot disease which devastates crops and their profitability. Particular attention is given to the link between soil and nutrient chemistry and activity of this microbe.

Differentiation in populations of the apple scab fungus Venturia inaequalis on cultivars in a mixed orchard remains over time

The ascomycete Venturia inaequalis causes annual epidemics of apple scab worldwide. Scab development is reduced in mixed cultivar orchards compared with monocultures. To use mixtures in commercial production, we need to understand how the population of scab changes in a mixed orchard and how likely a super race, with virulence factors overcoming multiple resistance factors in the mixed orchard, is to emerge and become dominant. We used short sequence repeat (SSR) markers to investigate the temporal change of scab populations in two mixed cultivar orchards in the UK to infer the likelihood of emergence of a scab super race. There were no significant differences between the populations at the two sampling times (six or seven years apart) in either of the two mixed orchards. In one of the orchards apple scab populations on different cultivars were significantly different and the differences did not diminish over time. These results suggest that it is not inevitable that a super race of V. inaequalis will become dominant during the lifetime of a commercial apple orchard.

The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

P>Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Delta ubc13 and Delta mms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.

Nuclear magnetic resonance characterization of metabolite disorder in orange trees caused by citrus sudden death disease

Citrus sudden death (CSD) is a new disease of sweet orange and mandarin trees grafted on Rangpur lime and Citrus volkameriana rootstocks. It was first seen in Brazil in 1999, and has since been detected in more than four million trees. The CSD causal agent is unknown and the current hypothesis involves a virus similar to Citrus tristeza virus or a new virus named Citrus sudden death-associated virus. CSD symptoms include generalized foliar discoloration, defoliation and root death, and, in most cases, it can cause tree death. One of the unique characteristics of CSD disease is the presence of a yellow stain in the rootstock bark near the bud union. This region also undergoes profound anatomical changes. In this study, we analyse the metabolic disorder caused by CSD in the bark of sweet orange grafted on Rangpur lime by nuclear magnetic resonance (NMR) spectroscopy and imaging. The imaging results show the presence of a large amount of non-functional phloem in the rootstock bark of affected plants. The spectroscopic analysis shows a high content of triacylglyceride and sucrose, which may be related to phloem blockage close to the bud union. We also propose that, without knowing the causal CSD agent, the determination of oil content in rootstock bark by low-resolution NMR can be used as a complementary method for CSD diagnosis, screening about 300 samples per hour.

Prácticas culturales para el manejo de la Sigatoka negra en bananos y plátanos

Black sigatoka (Mycosphaerella fijiensis) control consists in the continuous use of fungicide sprays and cultural practices. Cultural control reduces inoculum sources of the pathogen and favorable environmental conditions for its development, as well as it increases the vigor of the plants. In order to manage the disease it is necessary to know its behavior through the time, its relationship with the weather and the crop management. The most important cultural practice to reduce the inoculum source is the removal of the whole affected leaves or only the diseased portions of the leaves. The removed tissue is placed over the ground and the use of urea is feasible to accelerate its decomposition. An alternative practice is the "mini-composting" that consists in forming small heaps with the dead leaves and harvested plants for its quick degradation, which allows to reduce the pathogen inoculum and to incorporate nutrients and organic matters into the soil. The early pruning of the tips of the Young leaves before they show symptoms of the disease and the quick elimination of harvested plants reduce the inoculum. Also, the agronomic management of the crop helps to reduce the favorable conditions and to increase the vigor of the plants: this management considers plantation density, drainage systems, irrigation methods, control of weeds, chemical-biological fertilization, and nematode control.

Tamanho da amostra para quantificação do diâmetro de lesões de cancro cítrico

A estimativa do diâmetro de lesões de cancro cítrico é uma das principais técnicas usadas na avaliação da interação entre isolados x genótipos, na avaliação da resistência varietal e no estudo de aspectos epidemiológicos. No entanto, inexistem informações a respeito do tamanho da amostra para uma adequada quantificação da doença, considerando o diâmetro de lesões. Nesse sentido, o presente trabalho teve como objetivo a determinação do número de amostras para mensurar o diâmetro médio de lesões de cancro cítrico. Foram considerados como fontes de variação três genótipos do hospedeiro, dois métodos de inoculação de Xanthomonas citri subsp. citri e dois avaliadores. As avaliações dos diâmetros foram realizadas considerando ou não o halo amarelo ao redor do tecido necrosado, quando presente. Estimativas do diâmetro de lesões com erros na média inferiores a 3% são impraticáveis em razão do tamanho excessivo da amostra (mais que quarenta lesões/planta). Menores erros na média ocorreram nas estimativas considerando somente o tecido necrosado. Estimativas precisas do diâmetro de lesões, com erros na média inferiores a 10%, podem ser obtidas com tamanhos de amostras entre cinco e quinze lesões/planta, independentemente do genótipo e da idade das lesões.