A comparison between the role of enniatins and deoxynivalenol in Fusarium virulence on different tissues of common wheat.

BACKGROUND: Fusarium graminearum and Fusarium avenaceum are two of the most important causal agents of Fusarium head blight (FHB) of wheat. They can produce mycotoxins that accumulate in infected wheat heads, including deoxynivalenol (DON) and enniatins (ENNs), produced by F. graminearum and F. avenaceum, respectively. While the role of DON as a virulence factor in F. graminearum toward wheat is well known, ENNs in F. avenaceum has been poorly explored. Results obtained to-date indicate that ENNs may confer an advantage to F. avenaceum only on particular hosts. RESULTS: In this study, with the use of ENN-producing and ENN non-producing F. avenaceum strains, the role of ENNs on F. avenaceum virulence was investigated on the root, stem base and head of common wheat, and compared with the role of DON, using DON-producing and DON non-producing F. graminearum strains. The DON-producing F. graminearum strain showed a significantly higher ability to cause symptoms and colonise each of the tested tissues than the non-producing strain. On the other hand, the ability to produce ENNs increased initial symptoms of the disease and fungal biomass accumulation, measured by qPCR, only in wheat heads, and not in roots or stem bases. LC-MS/MS analysis was used to confirm the presence of ENNs and DON in the different strains, and results, both in vitro and in wheat heads, were consistent with the genetics of each strain. CONCLUSION: While the key role of DON on F. graminearum virulence towards three different wheat tissues was noticeable, ENNs seemed to have a role only in influencing F. avenaceum virulence on common wheat heads probably due to an initial delay in the appearance of symptoms.

Effect of the mycotoxins enniatin B and deoxynivalenol on the wheat aphid Sitobion avenae and on the predatory lacewing Chrysoperla carnea.

BACKGROUND: Fusarium species are responsible for Fusarium head blight (FHB) in wheat, resulting in yield losses and mycotoxin contamination. Deoxynivalenol (DON) and enniatins (ENNs) are common mycotoxins produced by Fusarium, affecting plant, animal and human health. Although DON's effects have been widely studied, limited research has explored the impact of ENNs on insects. This study examines the influence of DON and enniatin B (ENB), both singularly and in combination, on the wheat aphid Sitobion avenae and one of its predators, the lacewing Chrysoperla carnea. RESULTS: When exposed to DON (100 mg L-1) or DON + ENB (100 mg L-1), S. avenae exhibited significantly increased mortality compared to the negative control. ENB (100 mg L-1) had no significant effect on aphid mortality. DON-treated aphids showed increasing mortality from 48 to 96 h. A dose-response relationship with DON revealed significant cumulative mortality starting at 25 mg L-1. By contrast, C. carnea larvae exposed to mycotoxins via cuticular application did not show significant differences in mortality when mycotoxins were dissolved in water but exhibited increased mortality with acetone-solubilized DON + ENB (100 mg L-1). Feeding C. carnea with aphids exposed to mycotoxins (indirect exposure) did not impact their survival or predatory activity. Additionally, the impact of mycotoxins on C. carnea was observed only with acetone-solubilized DON + ENB. CONCLUSIONS: These findings shed light on the complex interactions involving mycotoxins, aphids and their predators, offering valuable insights for integrated pest management strategies. Further research should explore broader ecological consequences of mycotoxin contamination in agroecosystems. © 2024 Society of Chemical Industry.

Effect of Different Light Wavelengths on Zymoseptoria tritici Development and Leaf Colonization in Bread Wheat.

The wheat pathogen Zymoseptoria tritici can respond to light by modulating its gene expression. Because several virulence-related genes are differentially expressed in response to light, different wavelengths could have a crucial role in the Z. tritici-wheat interaction. To explore this opportunity, the aim of this study was to analyze the effect of blue (470 nm), red (627 nm), blue-red, and white light on the in vitro and in planta development of Z. tritici. The morphology (mycelium appearance, color) and phenotypic (mycelium growth) characteristics of a Z. tritici strain were evaluated after 14 days under the different light conditions in two independent experiments. In addition, bread wheat plants were artificially inoculated with Z. tritici and grown for 35 days under the same light treatments. The disease incidence, severity, and fungal DNA were analyzed in a single experiment. Statistical differences were determined by using an ANOVA. The obtained results showed that the different light wavelengths induced specific morphological changes in mycelial growth. The blue light significantly reduced colony growth, while the dark and red light favored fungal development (p < 0.05). The light quality also influenced host colonization, whereby the white and red light had stimulating and repressing effects, respectively (p < 0.05). This precursory study demonstrated the influence of light on Z. tritici colonization in bread wheat.

Impact of Enniatin and Deoxynivalenol Co-Occurrence on Plant, Microbial, Insect, Animal and Human Systems: Current Knowledge and Future Perspectives.

Fusarium mycotoxins commonly contaminate agricultural products resulting in a serious threat to both animal and human health. The co-occurrence of different mycotoxins in the same cereal field is very common, so the risks as well as the functional and ecological effects of mycotoxins cannot always be predicted by focusing only on the effect of the single contaminants. Enniatins (ENNs) are among the most frequently detected emerging mycotoxins, while deoxynivalenol (DON) is probably the most common contaminant of cereal grains worldwide. The purpose of this review is to provide an overview of the simultaneous exposure to these mycotoxins, with emphasis on the combined effects in multiple organisms. Our literature analysis shows that just a few studies on ENN-DON toxicity are available, suggesting the complexity of mycotoxin interactions, which include synergistic, antagonistic, and additive effects. Both ENNs and DON modulate drug efflux transporters, therefore this specific ability deserves to be explored to better understand their complex biological role. Additionally, future studies should investigate the interaction mechanisms of mycotoxin co-occurrence on different model organisms, using concentrations closer to real exposures.

Different diagnostic approaches for the characterization of the fungal community and Fusarium species complex composition of Italian durum wheat grain and correlation with secondary metabolite accumulation.

BACKGROUND: The evolution of the fungal communities associated with durum wheat was assessed using different diagnostic approaches. Durum wheat grain samples were collected in three different Italian cultivation macro-areas (north, center and south). Fungal isolation was realized by potato dextrose agar (PDA) and by deep-freezing blotter (DFB). Identification of Fusarium isolates obtained from PDA was achieved by partial tef1α sequencing (PDA + tef1α), while those obtained from DFB were identified from their morphological characteristics (DFB + mc). The fungal biomass of eight Fusarium species was quantified in grains by quantitative polymerase chain reaction (qPCR). Fungal secondary metabolites were analyzed in grains by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Correlations between Fusarium detection techniques (PDA + tef1α; DFB + mc and qPCR) and mycotoxins in grains were assessed. RESULTS: Alternaria and Fusarium showed the highest incidence among the fungal genera developed from grains. Within the Fusarium community, PDA + tef1α highlighted that F. avenaceum and F. graminearum were the most represented members, while, DFB + mc detected a high presence of F. proliferatum. Alternaria and Fusarium mycotoxins, principally enniatins, were particularly present in the grain harvested in central Italy. Deoxynivalenol was mainly detected in northern-central Italy. CONCLUSIONS: The adoption of the different diagnostic techniques of Fusarium detection highlighted that, for some species, qPCR was the best method of predicting their mycotoxin contamination in grains. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Infection timing affects Fusarium poae colonization of bread wheat spikes and mycotoxin accumulation in the grain.

BACKGROUND: Fusarium poae is one of the most common Fusarium head blight (FHB) causal agents in wheat. This species can biosynthesize a wide range of mycotoxins, in particular nivalenol (NIV). In FHB epidemiology, infection timing is important for disease occurrence, kernel development, symptom appearance and mycotoxin accumulation in grain. The present study explored, both in a controlled environment and in a 2-year field plot experiment in Central Italy, the influence of five infection timings (from beginning of flowering to medium milk growth stage) on F. poae colonization and mycotoxin accumulation in bread wheat spikes (spring cv. A416 and winter cv. Ambrogio). RESULTS: Both climate chamber and field experiments showed that early infection timings (from beginning of flowering to full flowering) especially favoured F. poae colonization and accumulation of its mycotoxins (particularly NIV) in grain. By contrast, later infection timings (watery ripe and medium milk) reduced F. poae development and mycotoxin levels. The time window of host susceptibility in the field was shorter than that observed under controlled conditions. Symptom expression in kernels also differed among infection timings. In general, F. poae biomass was higher in the chaff than in the grain. CONCLUSION: These results enhance knowledge of a common member of the FHB complex worldwide, and could be useful in forecasting the risk of F. poae infection and mycotoxin contamination. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Management of Pyrenophora teres f. teres, the Causal Agent of Net Form Net Blotch of Barley, in A Two-Year Field Experiment in Central Italy.

Pyrenophora teres is the causal agent of barley net blotch (NB), a disease that can be found in two different forms: net form (NFNB), caused by P. teres f. teres, and spot form (SFNB), caused by P. teres f. maculata. A two-year field experiment was carried out to evaluate the response to NB of six different barley cultivars for malt or feed/food production. In addition, the efficacy of several recently developed foliar fungicides with different modes of action (SDHI, DMI, and QoI) towards the disease was examined. After NB leaf symptom evaluation, the identification of P. teres forms was performed. Grain yield was determined, and pathogen biomass was quantified in the grain by qPCR. In the two experimental years characterized by different climatic conditions, only P. teres f. teres was detected. The tested cultivars showed different levels of NFNB susceptibility. In particular, the two-row cultivars for malt production showed the highest disease incidence. All applied fungicides exhibited a high efficacy in reducing disease symptoms on leaves and pathogen accumulation in grains. In fact, high levels of fungal biomass were detected only in the grain of the untreated malting barley cultivars. For some cultivars, grain yield was positively influenced by the application of fungicides.

Enniatin B and Deoxynivalenol Activity on Bread Wheat and on Fusarium Species Development.

Fusarium head blight (FHB) is a devastating wheat disease, mainly caused by Fusarium graminearum (FG)-a deoxynivalenol (DON)-producing species. However, Fusarium avenaceum (FA), able to biosynthesize enniatins (ENNs), has recently increased its relevance worldwide, often in co-occurrence with FG. While DON is a well-known mycotoxin, ENN activity, also in association with DON, is poorly understood. This study aims to explore enniatin B (ENB) activity, alone or combined with DON, on bread wheat and on Fusarium development. Pure ENB, DON, and ENB+DON (10 mg kg-1) were used to assess the impacts on seed germination, seedling growth, cell death induction (trypan blue staining), chlorophyll content, and oxidative stress induction (malondialdehyde quantification). The effect on FG and FA growth was tested using ENB, DON, and ENB+DON (10, 50, and 100 mg kg-1). Synergistic activity in the reduction of seed germination, growth, and chlorophyll degradation was observed. Conversely, antagonistic interaction in cell death and oxidative stress induction was found, with DON counteracting cellular stress produced by ENB. Fusarium species responded to mycotoxins in opposite directions. ENB inhibited FG development, while DON promoted FA growth. These results highlight the potential role of ENB in cell death control, as well as in fungal competition.

Phytopathological Threats Associated with Quinoa (Chenopodium quinoa Willd.) Cultivation and Seed Production in an Area of Central Italy.

In 2017, in a new Chenopodium quinoa cultivation area (Central Italy), emergence failures of the Titicaca, Rio Bamba, and Real varieties, whose seeds were obtained the previous year (2016) in the same location, were observed. Moreover, leaf disease symptoms on the Regalona variety, whose seeds came from Chile, were detected. Visual and microscopic analyses showed the presence of browning/necrotic symptoms on the seeds of the three varieties whose emergence in the field had failed. In addition, their in vitro germination rates were strongly compromised. Fusarium spp. was isolated with high incidence from Titicaca, Rio Bamba, and Real seeds. Among the detected Fusarium species, in the phylogenetic analysis, the dominant one clustered in the sub-clade Equiseti of the Fusarium incarnatum-equiseti (FIESC) species complex. Instead, the pathogen associated with Regalona leaf symptoms was identified, by morphological and molecular features, as Peronospora variabilis, the causal agents of downy mildew. This is the first report of both P. variabilis and F. equiseti on C. quinoa in Italy. Species-specific primers also detected P. variabilis in Regalona seeds. These results underline the importance of pathogen monitoring in new quinoa distribution areas, as well as of healthy seed production and import for successful cultivation.

In Vitro Evaluation of the Inhibitory Activity of Different Selenium Chemical Forms on the Growth of a Fusarium proliferatum Strain Isolated from Rice Seedlings.

In this study, the in vitro effects of different Se concentrations (5, 10, 15, 20, and 100 mg kg-1) from different Se forms (sodium selenite, sodium selenate, selenomethionine, and selenocystine) on the development of a Fusarium proliferatum strain isolated from rice were investigated. A concentration-dependent effect was detected. Se reduced fungal growth starting from 10 mg kg-1 and increasing the concentration (15, 20, and 100 mg kg-1) enhanced the inhibitory effect. Se bioactivity was also chemical form dependent. Selenocystine was found to be the most effective at the lowest concentration (5 mg kg-1). Complete growth inhibition was observed at 20 mg kg-1 of Se from selenite, selenomethionine, and selenocystine. Se speciation analysis revealed that fungus was able to change the Se speciation when the lowest Se concentration was applied. Scanning Electron Microscopy showed an alteration of the fungal morphology induced by Se. Considering that the inorganic forms have a higher solubility in water and are cheaper than organic forms, 20 mg kg-1 of Se from selenite can be suggested as the best combination suitable to inhibit F. proliferatum strain. The addition of low concentrations of Se from selenite to conventional fungicides may be a promising alternative approach for the control of Fusarium species.

Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum.

DNA methylation mediates organisms' adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

Fungicides may have differential efficacies towards the main causal agents of Fusarium head blight of wheat.

BACKGROUND: Fusarium head blight (FHB) is a complex disease of wheat and barley caused by several Fusarium species. In recent years, a variation in the composition of the FHB community has been observed in several wheat cultivation areas across the world. In detail, F. avenaceum and F. poae increased their frequencies, while, a lower F. graminearum and F. culmorum incidence was simultaneously observed. These shifts within the FHB complex might have been caused by different factors, including the selective pressure caused by fungicides used to control the disease in the field. Therefore, the present study was carried out to evaluate, both in in vitro experiments and in field trials, the activity of commonly used fungicides of wheat (tebuconazole, metconazole, prothioconazole and prochloraz) towards the above mentioned four Fusarium species. RESULTS: A preliminary in vitro assay revealed that low concentrations of all tested fungicides caused the incomplete reduction of fungal development. Furthermore, F. poae and F. avenaceum showed, at the same time, a lower sensitivity to all tested fungicides. In field trials, all fungicides showed an activity against the four Fusarium species. However, F. avenaceum exhibited a reduced sensitivity to metconazole. The lower efficacy of metconazole towards F. avenaceum was also confirmed by an additional in vitro experiment on several F. avenaceum and F. graminearum different strains. CONCLUSION: The selective pressure exerted by the extensive use of certain fungicides may influence population dynamics of Fusarium species due to their different sensitivity. © 2020 Society of Chemical Industry.

Cultivation Area Affects the Presence of Fungal Communities and Secondary Metabolites in Italian Durum Wheat Grains.

In this study, durum wheat kernels harvested in three climatically different Italian cultivation areas (Emilia Romagna, Umbria and Sardinia) in 2015, were analyzed with a combination of different isolation methods to determine their fungal communities, with a focus on Fusarium head blight (FHB) complex composition, and to detect fungal secondary metabolites in the grains. The genus Alternaria was the main component of durum wheat mycobiota in all investigated regions, with the Central Italian cultivation area showing the highest incidence of this fungal genus and of its secondary metabolites. Fusarium was the second most prevalent genus of the fungal community in all cultivation environments, even if regional differences in species composition were detected. In particular, Northern areas showed the highest Fusarium incidence, followed by Central and then Southern cultivation areas. Focusing on the FHB complex, a predominance of Fusariumpoae, in particular in Northern and Central cultivation areas, was found. Fusariumgraminearum, in the analyzed year, was mainly detected in Emilia Romagna. Because of the highest Fusarium incidence, durum wheat harvested in the Northern cultivation area showed the highest presence of Fusarium secondary metabolites. These results show that durum wheat cultivated in Northern Italy may be subject to a higher FHB infection risk and to Fusarium mycotoxins accumulation.

Effect of wheat infection timing on Fusarium head blight causal agents and secondary metabolites in grain.

Fusarium head blight (FHB) results in yield loss and damaging contamination of cereal grains and can be caused by several Fusarium species. The objective of the present study was to determine, in a greenhouse experiment on winter wheat, how FHB was affected by timing of infection (0, 3, 6 or 9 days after anthesis, daa) by the aggressive species Fusarium graminearum compared to the relatively weak species Fusarium avenaceum, Fusarium poae and Fusarium acuminatum. Measures of FHB development were: symptoms in spikes (visually assessed), fungal biomass (quantified by real time quantitative PCR) and accumulation of fungal secondary metabolites (quantified by liquid chromatography-tandem mass spectrometry) in kernels. With regard to symptoms, F. graminearum was unaffected by inoculation timing, while the weaker pathogens caused greater disease severity at later timings. In contrast, the accumulation of F. graminearum biomass was strongly affected by inoculation timing (3 daa ≥ 6 daa ≥ 0 daa = 9 daa), while colonization by the weaker pathogens was less influenced. Similarly, F. graminearum secondary metabolite accumulation was affected by inoculation timing (3 daa ≥ 6 daa ≥ 0 daa = 9 daa), while that of the weaker species was less affected. However, secondary metabolites produced by these weaker species tended to be higher from intermediate-late inoculations (6 daa). Overall, infection timing appeared to play a role particularly in F. graminearum colonization and secondary metabolite accumulation. However, secondary metabolites of weaker Fusarium species may be relatively more abundant when environmental conditions promote spore dispersal later in anthesis, while secondary metabolites produced by F. graminearum are relatively favored by earlier conducive conditions.

Fungal community, Fusarium head blight complex and secondary metabolites associated with malting barley grains harvested in Umbria, central Italy.

In recent years, due to the negative impact of toxigenic mycobiota and of the accumulation of their secondary metabolites in malting barley grains, monitoring the evolution of fungal communities in a certain cultivation area as well as detecting the different mycotoxins present in the raw material prior to malting and brewing processes have become increasingly important. In this study, a survey was carried out on malting barley samples collected after their harvest in the Umbria region (central Italy). Samples were analyzed to determine the composition of the fungal community, to identify the isolated Fusarium species, to quantify fungal secondary metabolites in the grains and to characterize the in vitro mycotoxigenic profile of a subset of the isolated Fusarium strains. The fungal community of barley grains was mainly composed of microorganisms belonging to the genus Alternaria (77%), followed by those belonging to the genus Fusarium (27%). The Fusarium head blight (FHB) complex was represented by nine species with the predominance of Fusarium poae (37%), followed by Fusarium avenaceum (23%), Fusarium graminearum (22%) and Fusarium tricinctum (7%). Secondary metabolites biosynthesized by Alternaria and Fusarium species were present in the analyzed grains. Among those biosynthesized by Fusarium species, nivalenol and enniatins were the most prevalent ones. Type A trichothecenes (T-2 and HT-2 toxins) as well as beauvericin were also present with a high incidence. Conversely, the number of samples contaminated with deoxynivalenol was low. Conjugated forms, such as deoxynivalenol-3-glucoside and HT-2-glucoside, were detected for the first time in malting barley grains cultivated in the surveyed area. In addition, strains of F. avenaceum and F. tricinctum showed the ability to biosynthesize in vitro high concentrations of enniatins. The analysis of fungal secondary metabolites, both in the grains and in vitro, revealed also the presence of other compounds, for which further investigations will be required. The combination of microbiological analyses, of molecular biology assays and of multi-mycotoxin screening shed light on the complexity of the fungal community and its secondary metabolites released in malting barley.

Changes in the Fusarium Head Blight Complex of Malting Barley in a Three-Year Field Experiment in Italy.

In this study, conducted for three years on eleven malting barley varieties cultivated in central Italy, the incidence of different mycotoxigenic fungal genera, the identification of the Fusarium species associated with the Fusarium Head Blight (FHB) complex, and kernels contamination with deoxynivalenol (DON) and T-2 mycotoxins were determined. The influence of climatic conditions on Fusarium infections and FHB complex composition was also investigated. Fusarium species were always present in the three years and the high average and maximum temperatures during anthesis mainly favored their occurrence. The FHB complex was subject to changes during the three years and the main causal agents were F. poae, F. avenaceum, F. tricinctum and F. graminearum, which, even if constantly present, never represented the principal FHB agent. The relative incidence of Fusarium species changed because of climatic conditions occurring during the seasons. The FHB complex was composed of many different Fusarium species and some of them were associated with a specific variety and/or with specific weather parameters, indicating that the interaction between a certain plant genotype and climatic conditions may influence the presence of Fusarium spp. causing infections. With regard to mycotoxin contamination, T-2 toxin, in some cases, was found in kernels at levels that exceeded EU recommended values.

Presence of Fusarium Species and Other Toxigenic Fungi in Malting Barley and Multi-Mycotoxin Analysis by Liquid Chromatography-High-Resolution Mass Spectrometry.

A study was carried out on 43 malting barley samples collected in 2013 across the Umbria region (central Italy) to determine the incidence of the principal mycotoxigenic fungal genera, to identify the Fusarium species isolated from the grains, and to detect the presence of 34 fungal secondary metabolites by liquid chromatography-high-resolution mass spectrometry. The multimycotoxin-method development involved the evaluation of both a two-step solvent and QuEChERS protocol for metabolite extraction. The former protocol was selected because of better accuracy, which was evaluated on the basis of spike-recovery experiments. The most frequently isolated fungal species belonged to the genera Alternaria and Fusarium. The predominant Fusarium species was F. avenaceum, followed by F. graminearum. HT-2 toxin was the most frequently detected mycotoxin, followed by enniatin B, enniatin B1, T-2 toxin, and nivalenol. As a consequence of the observed mixed fungal infections, mycotoxin co-occurrence was also detected. A combination of mycological and mycotoxin analyses allowed the ability to obtain comprehensive information about the presence of mycotoxigenic fungi and their contaminants in malting barley cultivated in a specific geographic area.

EffectorP: predicting fungal effector proteins from secretomes using machine learning.

Eukaryotic filamentous plant pathogens secrete effector proteins that modulate the host cell to facilitate infection. Computational effector candidate identification and subsequent functional characterization delivers valuable insights into plant-pathogen interactions. However, effector prediction in fungi has been challenging due to a lack of unifying sequence features such as conserved N-terminal sequence motifs. Fungal effectors are commonly predicted from secretomes based on criteria such as small size and cysteine-rich, which suffers from poor accuracy. We present EffectorP which pioneers the application of machine learning to fungal effector prediction. EffectorP improves fungal effector prediction from secretomes based on a robust signal of sequence-derived properties, achieving sensitivity and specificity of over 80%. Features that discriminate fungal effectors from secreted noneffectors are predominantly sequence length, molecular weight and protein net charge, as well as cysteine, serine and tryptophan content. We demonstrate that EffectorP is powerful when combined with in planta expression data for predicting high-priority effector candidates. EffectorP is the first prediction program for fungal effectors based on machine learning. Our findings will facilitate functional fungal effector studies and improve our understanding of effectors in plant-pathogen interactions. EffectorP is available at http://effectorp.csiro.au.