MinION Sequencing of Fungi in Sub-Saharan African Air and a Novel LAMP Assay for Rapid Detection of the Tropical Phytopathogenic Genus Lasiodiplodia.

To date, there have been no DNA-based metabarcoding studies into airborne fungi in tropical Sub-Saharan Africa. In this initial study, 10 air samples were collected onto Vaseline-coated acrylic rods mounted on drones flown at heights of 15-50 meters above ground for 10-15 min at three sites in Ghana. Purified DNA was extracted from air samples, the internal transcribed spacer (ITS) region was amplified using fungal-specific primers, and MinION third-generation amplicon sequencing was undertaken with downstream bioinformatics analyses utilizing GAIA cloud-based software (at genus taxonomic level). Principal coordinate analyses based on Bray-Curtis beta diversity dissimilarity values found no clear evidence for the structuring of fungal air communities, nor were there significant differences in alpha diversity, based on geographic location (east vs. central Ghana), underlying vegetation type (cocoa vs. non-cocoa), or height above ground level (15-23 m vs. 25-50 m), and despite the short flight times (10-15 min), ~90 operational taxonomic units (OTUs) were identified in each sample. In Ghanaian air, fungal assemblages were skewed at the phylum taxonomic level towards the ascomycetes (53.7%) as opposed to basidiomycetes (24.6%); at the class level, the Dothideomycetes were predominant (29.8%) followed by the Agaricomycetes (21.8%). The most common fungal genus in Ghanaian air was cosmopolitan and globally ubiquitous Cladosporium (9.9% of reads). Interestingly, many fungal genera containing economically important phytopathogens of tropical crops were also identified in Ghanaian air, including Corynespora, Fusarium, and Lasiodiplodia. Consequently, a novel loop-mediated isothermal amplification (LAMP) assay, based on translation elongation factor-1α sequences, was developed and tested for rapid, sensitive, and specific detection of the fungal phytopathogenic genus Lasiodiplodia. Potential applications for improved tropical disease management are considered.

Efficient qPCR estimation and discrimination of airborne inoculum of Leptosphaeria maculans and L. biglobosa, the causal organisms of phoma leaf spotting and stem canker of oilseed rape.

BACKGROUND: Detection of the inoculum of phytopathogens greatly assists in the management of diseases, but is difficult for pathogens with airborne fungal propagules. Here, we present experiments to determine the abundance and distribution frequencies of the ascospores of Leptosphaeria (Plenodomus) species that were collected on the tapes of volumetric Hirst-type traps near oilseed rape fields in Poznan, Poland and Harpenden, UK. Fungal detection and species discrimination were achieved using a SYBR-Green quantitative polymerase chain reaction (qPCR) with two different pairs of primers previously reported to differentiate Leptosphaeria maculans (Plenodomus lingam) or L. biglobosa (P. biglobosus). RESULTS: Detection was successful even at fewer than five spores per m3 of air. The primer pairs differed in the correlation coefficients obtained between DNA yields and the daily abundance of ascospores that were quantified by microscopy on duplicate halves of the spore trap tapes. Important differences in the specificity and sensitivity of the published SYBR-Green assays were also found, indicating that the Liu primers did not detect L. biglobosa subclade 'canadensis', whereas the Mahuku primers detected L. biglobosa subclade 'canadensis' and also the closely related Plenodomus dezfulensis. CONCLUSIONS: Comparisons confirmed that application of qPCR assays to spore trap samples can be used for the early detection, discrimination and quantification of aerially dispersed L. maculans and L. biglobosa propagules before leaf spot symptoms are visible in winter oilseed rape fields. The specificity of the primers must be taken into consideration because the final result will greatly depend on the local population of the pathogen. © 2023 Society of Chemical Industry.

Indirect Evidence Based on Mating-Type Ratios for the Role of Sexual Reproduction in European and Chinese Populations of Plenodomus biglobosus (Blackleg of Oilseed Rape).

Blackleg (Phoma) disease, caused by the ascomycete fungi Plenodomus biglobosus and P. lingam, threatens oilseed rape (OSR; Brassica napus) crops internationally. In many parts of the world, both species co-occur, but in China only P. biglobosus has so far been reported. Plenodomus biglobosus reproduces asexually (pycnidiospores), but also sexually (pseudothecia-yielding ascospores), via a heterothallic mating system requiring MAT1-1 and MAT1-2 genotypes. However, the roles of airborne ascospore inoculum in driving blackleg disease outbreaks in China are less well understood compared to elsewhere in the world. This is despite the very different agronomic cropping practices in parts of China, in which paddy rice and OSR are often grown in rotation; OSR stubble is often submerged under water for long periods potentially affecting pseudothecial development. Here, we indirectly investigate the potential role of sexual reproduction by developing new polymerase chain reaction (PCR) -based mating-type diagnostics for P. biglobosus and subsequently screening an international collection of 59 European and 157 Chinese isolates. Overall, in both Europe and China, P. biglobosus mating types did not deviate from a 1:1 ratio, such as is generally thought to occur under frequency-dependent selection in sexually reproducing pathogen populations. Both mating types were balanced in all the individual European countries tested (Austria, France, Poland, UK). Conversely, in China, mating types were only balanced in the eastern region; in the northern and southwestern regions there were skewed ratios, more typical of predominantly asexual reproduction, towards MAT1-1 and MAT1-2, respectively. The implications of these findings and future research directions for improved understanding of P. biglobosus epidemiology on OSR, particularly in China, are considered.

Take-All Disease: New Insights into an Important Wheat Root Pathogen.

Take-all disease, caused by the fungal root pathogen Gaeumannomyces tritici, is considered to be the most important root disease of wheat worldwide. Here we review the advances in take-all research over the last 15 years, focusing on the identification of new sources of genetic resistance in wheat relatives and the role of the microbiome in disease development. We also highlight recent breakthroughs in the molecular interactions between G. tritici and wheat, including genome and transcriptome analyses. These new findings will aid the development of novel control strategies against take-all disease. In light of this growing understanding, the G. tritici-wheat interaction could provide a model study system for root-infecting fungal pathogens of cereals.

Novel Multiplex and Loop-Mediated Isothermal Amplification Assays for Rapid Species and Mating-Type Identification of Oculimacula acuformis and O. yallundae (Causal Agents of Cereal Eyespot), and Application for Detection of Ascospore Dispersal and In Planta Use.

Eyespot, caused by the related fungal pathogens Oculimacula acuformis and O. yallundae, is an important cereal stem-base disease in temperate parts of the world. Both species are dispersed mainly by splash-dispersed conidia but are also known to undergo sexual reproduction, yielding apothecia containing ascospores. Field diagnosis of eyespot can be challenging, with other pathogens causing similar symptoms, which complicates eyespot management strategies. Differences between O. acuformis and O. yallundae (e.g., host pathogenicity and fungicide sensitivity) require that both be targeted for effective disease management. Here, we develop and apply two molecular methods for species-specific and mating-type (MAT1-1 or MAT1-2) discrimination of O. acuformis and O. yallundae isolates. First, a multiplex PCR-based diagnostic assay targeting the MAT idiomorph region was developed, allowing simultaneous determination of both species and mating type. This multiplex PCR assay was successfully applied to type a global collection of isolates. Second, the development of loop-mediated isothermal amplification (LAMP) assays targeting ß-tubulin sequences, which allow fast (<9 min) species-specific discrimination of global O. acuformis and O. yallundae isolates, is described. The LAMP assay can detect very small amounts of target DNA (1 pg) and was successfully applied in planta. In addition, mating-type-specific LAMP assays were also developed for rapid (<12 min) genotyping of O. acuformis and O. yallundae isolates. Finally, the multiplex PCR-based diagnostic was applied, in conjunction with spore trapping in field experiments, to provide evidence of the wind dispersal of ascospores from a diseased crop. The results indicate an important role of the sexual cycle in the dispersal of eyespot.

Characterization of an antimicrobial and phytotoxic ribonuclease secreted by the fungal wheat pathogen Zymoseptoria tritici.

The fungus Zymoseptoria tritici is the causal agent of Septoria Tritici Blotch (STB) disease of wheat leaves. Zymoseptoria tritici secretes many functionally uncharacterized effector proteins during infection. Here, we characterized a secreted ribonuclease (Zt6) with an unusual biphasic expression pattern. Transient expression systems were used to characterize Zt6, and mutants thereof, in both host and non-host plants. Cell-free protein expression systems monitored the impact of Zt6 protein on functional ribosomes, and in vitro assays of cells treated with recombinant Zt6 determined toxicity against bacteria, yeasts and filamentous fungi. We demonstrated that Zt6 is a functional ribonuclease and that phytotoxicity is dependent on both the presence of a 22-amino-acid N-terminal 'loop' region and its catalytic activity. Zt6 selectively cleaves both plant and animal rRNA species, and is toxic to wheat, tobacco, bacterial and yeast cells, but not to Z. tritici itself. Zt6 is the first Z. tritici effector demonstrated to have a likely dual functionality. The expression pattern of Zt6 and potent toxicity towards microorganisms suggest that, although it may contribute to the execution of wheat cell death, it is also likely to have an important secondary function in antimicrobial competition and niche protection.

Fungal Communities Including Plant Pathogens in Near Surface Air Are Similar across Northwestern Europe.

Information on the diversity of fungal spores in air is limited, and also the content of airborne spores of fungal plant pathogens is understudied. In the present study, a total of 152 air samples were taken from rooftops at urban settings in Slagelse, DK, Wageningen NL, and Rothamsted, UK together with 41 samples from above oilseed rape fields in Rothamsted. Samples were taken during 10-day periods in spring and autumn, each sample representing 1 day of sampling. The fungal content of samples was analyzed by metabarcoding of the fungal internal transcribed sequence 1 (ITS1) and by qPCR for specific fungi. The metabarcoding results demonstrated that season had significant effects on airborne fungal communities. In contrast, location did not have strong effects on the communities, even though locations were separated by up to 900 km. Also, a number of plant pathogens had strikingly similar patterns of abundance at the three locations. Rooftop samples were more diverse than samples taken above fields, probably reflecting greater mixing of air from a range of microenvironments for the rooftop sites. Pathogens that were known to be present in the crop were also found in air samples taken above the field. This paper is one of the first detailed studies of fungal composition in air with the focus on plant pathogens and shows that it is possible to detect a range of pathogens in rooftop air samplers using metabarcoding.

Novel Technologies for the detection of Fusarium head blight disease and airborne inoculum.

Many pathogens are dispersed by airborne spores, which can vary in space and time. We can use air sampling integrated with suitable diagnostic methods to give a rapid warning of inoculum presence to improve the timing of control options, such as fungicides. Air sampling can also be used to monitor changes in genetic traits of pathogen populations such as the race structure or frequency of fungicide resistance. Although some image-analysis methods are possible to identify spores, in many cases, species-specific identification can only be achieved by DNA-based methods such as qPCR and LAMP and in some cases by antibody-based methods (lateral flow devices) and biomarker-based methods ('electronic noses' and electro-chemical biosensors). Many of these methods also offer the prospect of rapid on-site detection to direct disease control decisions. Thresholds of spore concentrations that correspond to a disease risk depend on the sampler (spore-trap) location (whether just above the crop canopy, on a UAV or drone, or on a tall building) and also need to be considered with weather-based infection models. Where disease control by spore detection is not possible, some diseases can be detected at early stages using optical sensing methods, especially chlorophyll fluorescence. In the case of Fusarium infections on wheat, it is possible to map locations of severe infections, using optical sensing methods, to segregate harvesting of severely affected areas of fields to avoid toxins entering the food chain. This is most useful where variable crop growth or microclimates within fields generate spatially variable infection, i.e. parts of fields that develop disease, while other areas have escaped infection and do not develop any disease.

Apoplastic recognition of multiple candidate effectors from the wheat pathogen Zymoseptoria tritici in the nonhost plant Nicotiana benthamiana.

The fungus Zymoseptoria tritici is a strictly apoplastic, host-specific pathogen of wheat leaves and causal agent of septoria tritici blotch (STB) disease. All other plants are considered nonhosts, but the mechanism of nonhost resistance (NHR) to Z. tritici has not been addressed previously. We sought to develop Nicotiana benthamiana as a system to study NHR against Z. tritici. Fluorescence microscopy and quantitative reverse transcription polymerase chain reactions were used to establish the interaction between Z. tritici and N. benthamiana. Agrobacterium-mediated transient expression was used to screen putative Z. tritici effector genes for recognition in N. benthamiana, and virus-induced gene silencing (VIGS) was employed to determine the role of two receptor-like kinases (RLKs), NbBAK1 and NbSOBIR1, in Z. tritici effector recognition. Numerous Z. tritici putative effectors (14 of 63 tested) induced cell death or chlorosis in N. benthamiana. For most, phenotypes were light-dependent and required effector secretion to the leaf apoplastic space. Moreover, effector-induced host cell death was dependent on NbBAK1 and NbSOBIR1. Our results indicate widespread recognition of apoplastic effectors from a wheat-infecting fungal pathogen in a taxonomically distant nonhost plant species presumably by cell surface immune receptors. This suggests that apoplastic recognition of multiple nonadapted pathogen effectors may contribute to NHR.

Pseudomonas spp. diversity is negatively associated with suppression of the wheat take-all pathogen.

Biodiversity and ecosystem functioning research typically shows positive diversity- productivity relationships. However, local increases in species richness can increase competition within trophic levels, reducing the efficacy of intertrophic level population control. Pseudomonas spp. are a dominant group of soil bacteria that play key roles in plant growth promotion and control of crop fungal pathogens. Here we show that Pseudomonas spp. richness is positively correlated with take-all disease in wheat and with yield losses of ~3 t/ha in the field. We modeled the interactions between Pseudomonas and the take-all pathogen in abstract experimental microcosms, and show that increased bacterial genotypic richness escalates bacterial antagonism and decreases the ability of the bacterial community to inhibit growth of the take-all pathogen. Future work is required to determine the generality of these negative biodiversity effects on different media and directly at infection zones on root surfaces. However, the increase in competition between bacteria at high genotypic richness and the potential loss of fungal biocontrol activity highlights an important mechanism to explain the negative Pseudomonas diversity-wheat yield relationship we observed in the field. Together our results suggest that the effect of biodiversity on ecosystem functioning can depend on both the function and trophic level of interest.

The induction of mycotoxins by trichothecene producing Fusarium species.

In recent years, many Fusarium species have emerged which now threaten the productivity and safety of small grain cereal crops worldwide. During floral infection and post-harvest on stored grains the Fusarium hyphae produce various types of harmful mycotoxins which subsequently contaminate food and feed products. This article focuses specifically on the induction and production of the type B sesquiterpenoid trichothecene mycotoxins. Methods are described which permit in liquid culture the small or large scale production and detection of deoxynivalenol (DON) and its various acetylated derivatives. A wheat (Triticum aestivum L.) ear inoculation assay is also explained which allows the direct comparison of mycotoxin production by species, chemotypes and strains with different growth rates and/or disease-causing abilities. Each of these methods is robust and can be used for either detailed time-course studies or end-point analyses. Various analytical methods are available to quantify the levels of DON, 3A-DON and 15A-DON. Some criteria to be considered when making selections between the different analytical methods available are briefly discussed.

A combined ¹H nuclear magnetic resonance and electrospray ionization-mass spectrometry analysis to understand the basal metabolism of plant-pathogenic Fusarium spp.

Many ascomycete Fusarium spp. are plant pathogens that cause disease on both cereal and noncereal hosts. Infection of wheat ears by Fusarium graminearum and F. culmorum typically results in bleaching and a subsequent reduction in grain yield. Also, a large proportion of the harvested grain can be spoiled when the colonizing Fusarium mycelia produce trichothecene mycotoxins, such as deoxynivalenol (DON). In this study, we have explored the intracellular polar metabolome of Fusarium spp. in both toxin-producing and nonproducing conditions in vitro. Four Fusarium spp., including nine well-characterized wild-type field isolates now used routinely in laboratory experimentation, were explored. A metabolic "triple-fingerprint" was recorded using (1)H nuclear magnetic resonance and direct-injection electrospray ionization-mass spectroscopy in both positive- and negative-ionization modes. These combined metabolomic analyses revealed that this technique is sufficient to resolve different wild-type isolates and different growth conditions. Principal components analysis was able to resolve the four species explored-F. graminearum, F. culmorum, F. pseudograminearum, and F. venenatum-as well as individual isolate differences from the same species. The external nutritional environment was found to have a far greater influence on the metabolome than the genotype of the organism. Conserved responses to DON-inducing medium were evident and included increased abundance of key compatible solutes, such as glycerol and mannitol. In addition, the concentration of γ-aminobutyric acid was elevated, indicating that the cellular nitrogen status may be affected by growth on DON-inducing medium.