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Background: Maple is an important ornamental plant in China. With the increasing use of maple trees in landscaping, a symptom of shoot dieback has been observed in Henan province, China., Results: In this study, 28 Diaporthe isolates were obtained from symptomatic shoots of maple trees between 2020 and 2023. Phylogenetic analyses based on five loci (ITS, TEF, CAL, HIS and TUB) coupled with morphology of 12 representative isolates identified three known species (D. eres, D. pescicola and D. spinosa) and one new species, namely D. pseudoacerina sp. nov. Koch's postulates confirmed that all these species were pathogenic. Additionally, D. pseudoacerina was able to infect China wingnut (Pterocarya stenoptera), pear (Pyrus sp.), and black locust (Robinia pseudoacacia). This study marks the first report of Diaporthe spinosa and D. pescicola pathogens infecting maple trees., Conclusions: These findings enhance the existing knowledge of the taxonomy and host diversity of Diaporthe species as, while also providing valuable information for managing of maple shoot dieback in Henan Province, China., (© 2024. The Author(s).)

Cotton (Gossypium hirsutum, Malvaceae) is the most important fiber crop in the world. There are published records of many fungal pathogens attacking Gossypium spp., causing numerous diseases, including powdery mildews. Recently, in 2022, non-cultivated spontaneous G. hirsutum plants bearing powdery mildews symptoms were found at roadsides in two municipalities of the state of Minas Gerais (Brazil): Varginha and Ubá. Such localities are situated ca. 260 km apart, suggesting a broader distribution of this fungus-host association in Brazil. Samples were taken to the laboratory, and an Ovulariopsis-like, asexual stage of Phyllactinia, was identified forming amphigenous colonies, that were more evident, white and cottony, abaxially. Morphological and molecular data- of the ITS and LSU regions- have shown that colonies from those two samples were of the same fungus species, belonging to a previously unknown species of Erysiphaceae (Ascomycota). The fungus fits into the Phyllactinia clade and is described herein as the new species Phyllactinia gossypina sp. nov. This new species belongs to the 'basal Phyllactinia group', a lineage that includes species known only from the Americas. This report expands the list of pathogenic fungi on cotton. It is early to anticipate whether this new powdery mildew represents a threat to cultivated cotton, which is a major crop in Brazil. Nevertheless, further studies about its infectivity to commercial cotton varieties are recommended, since all known Erysiphaceae are specialized obligate plant parasites and several species cause major losses to important crops., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Botryosphaeriaceae species are the major causal agents of walnut dieback worldwide, along with Diaporthe species. Botryosphaeria dothidea and Neofusicoccum parvum are the only two Botryosphaeriaceae species associated with this recently emergent disease in France, and little is known about their diversity, structure, origin and dispersion in French walnut orchards. A total of 381 isolates of both species were genetically typed using a sequence-based microsatellite genotyping (SSR-seq) method. This analysis revealed a low genetic diversity and a high clonality of these populations, in agreement with their clonal mode of reproduction. The genetic similarity among populations, regardless of the tissue type and the presence of symptoms, supports the hypothesis that these pathogens can move between fruits and twigs and display latent pathogen lifestyles. Contrasting genetic patterns between N. parvum populations from Californian and Spanish walnut orchards and the French ones suggested no conclusive evidence for pathogen transmission from infected materials. The high genetic similarity with French vineyards populations suggested instead putative transmission between these hosts, which was also observed with B. dothidea populations. Overall, this study provides critical insight into the epidemiology of two important pathogens involved in the emerging dieback of French walnut orchards, including their distribution, potential to mate, putative origin and disease pathways., (© 2024. The Author(s).)

Chestnut production is considered one of the most important economic resources of rural mountainous areas in Greece. Lately, producers report a steep rise in the incidence of brown rot disease caused by the fungus Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales), which results in severe chestnut rot. The pathogen is considered an emerging pathogen in many countries worldwide (Italy, France, Switzerland, Australia, New Zealand). This study aimed at (a) exploring the incidence of the brown rot disease in Vria (Regional Unit of Pieria, Region of Central Makedonia, Greece), (b) isolating and identifying the causal agent of the disease, (c) exploring the fungus presence at different phenological stages of the chestnut trees, and (d) implementing species-specific Bar- High Resolution Melting Analysis (HRM) for the early detection of G. smithogilvyi in chestnuts. G. smithogilvyi occurrence in chestnut tissues was more severe in June (59 %), nearly disappeared in July (19 %) and August (7 %) and increased again during harvesting time in September (57 %). This result could be attributed to a sum of different factors, including climate conditions. Moreover, it was demonstrated that G. smithogilvyi can be identified using a Bar-HRM analysis of chestnut tissues (buds, flowers and nuts). Results of this study clearly demonstrate that Bar-HRM can be used for the accurate, rapid and reliable identification of G. smithogilvyi universally on infected samples from different localities., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Eucalyptus spp. are undoubtedly one of the most favored plantation trees globally. Accurately identifying Eucalyptus pathogens is therefore crucial for timely disease prevention and control. Recently, symptoms of a leaf blight disease were observed on Eucalyptus trees in plantations at Jhajjar and Karnal in the state of Haryana, northern India. Asexual morphs resembling the features of the Botryosphaeriaceae were consistently isolated from the symptomatic leaves. Morphological features coupled with DNA sequence analysis confirmed a novel species, which is described and illustrated here as Botryosphaeria eucalypti sp. nov. Conidia of the new taxon are longer and wider than those of its phylogenetic neighbors. A distinct phylogenetic position for the new taxon was established through combined analysis of the internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1) and partial β-tubulin (tub2) regions. Recombination analysis provided additional support for the new species hypothesis. The pathogenicity of the novel species was proved on Eucalyptus leaves, and Koch's postulates were fulfilled. The discovery of new Botryosphaeria species is important because it will help in understanding the species diversity, host range, possible threats and disease control in the long run., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Field peas are grown by smallholder farmers in Ethiopia for food, fodder, income, and soil fertility. However, leaf diseases such as ascochyta blight, powdery mildew, and leaf spots affect the quantity and quality of this crop as well as crop growth. Experts use visual observation to detect field pea disease. However, this approach is expensive, labor-intensive, and imprecise. Therefore, in this study, we presented a transfer learning approach for the automatic diagnosis of field pea leaf diseases. We classified three field pea leaf diseases: Ascochyta blight, leaf spot, and powdery mildew. A softmax classifier was used to classify the diseases. A total of 1600 images of both healthy and diseased leaves were used to train, validate, and test the pretrained models. According to the experimental results, DenseNet121 achieved 99.73% training accuracy, 99.16% validation accuracy, and 98.33% testing accuracy after 100 epochs. we expect that this research work will offer various benefits for farmers and farm experts. It reduced the cost and time needed for the detection and classification of field pea leaf disease. Thus, a fast, automated, less costly, and accurate detection method is necessary to overcome the detection problem., Competing Interests: NO authors have competing interests, (Copyright: © 2024 Girmaw, Muluneh. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Field surveys conducted during 2021 and 2022 in Western Sicily, Italy, revealed the presence of common fig trees severely affected by trunk and crown root canker and bark cracking. Moreover, in conjunction with the symptomatic tissues, the same surveyed plants showed the presence of bark beetle holes and internal wood galleries. The predominant beetle Criphalus dilutus was previously reported attacking figs in Sicily. Phylogenetic analyses based on multilocus DNA data showed the presence of different fungal taxa associated with disease symptoms, including Botryosphaeria dothidea , Ceratocystis ficicola , Diaporthe foeniculina , Neocosmospora bostrycoides , N. perseae , and Neofusicoccum luteum . Pathogenicity tests conducted on potted fig plants showed that all the species were pathogenic to fig, with C. ficicola and Neocosmospora spp. as the most aggressive fungal species. Moreover, isolations conducted from the bodies of emerging adult insects recovered from disease samples confirmed the presence of C. ficicola and Neocosmospora spp., suggesting the potential involvement of C. dilutus in their dissemination., Competing Interests: The author(s) declare no conflict of interest.

Dynamic transposition of transposable elements (TEs) in fungal pathogens has significant impact on genome stability, gene expression, and virulence to the host. In Magnaporthe oryzae , genome plasticity resulting from TE insertion is a major driving force leading to the rapid evolution and diversification of this fungus. Despite their importance in M. oryzae population evolution and divergence, our understanding of TEs in this context remains limited. Here, we conducted a genome-wide analysis of TE transposition dynamics in the 11 most abundant TE families in M. oryzae populations. Our results show that these TEs have specifically expanded in recently isolated M. oryzae rice populations, with the presence/absence polymorphism of TE insertions highly concordant with population divergence on Geng/ Japonica and Xian/ Indica rice cultivars. Notably, the genes targeted by clade-specific TEs showed clade-specific expression patterns and are involved in the pathogenic process, suggesting a transcriptional regulation of TEs on targeted genes. Our study provides a comprehensive analysis of TEs in M. oryzae populations and demonstrates a crucial role of recent TE bursts in adaptive evolution and diversification of the M. oryzae rice-infecting lineage., Importance: Magnaporthe oryzae is the causal agent of the destructive blast disease, which caused massive loss of yield annually worldwide. The fungus diverged into distinct clades during adaptation toward the two rice subspecies, Xian/ Indica and Geng/ Japonica . Although the role of TEs in the adaptive evolution was well established, mechanisms underlying how TEs promote the population divergence of M. oryzae remain largely unknown. In this study, we reported that TEs shape the population divergence of M. oryzae by differentially regulating gene expression between Xian/ Indica -infecting and Geng/ Japonica -infecting populations. Our results revealed a TE insertion-mediated gene expression adaption that led to the divergence of M. oryzae population infecting different rice subspecies., Competing Interests: The authors declare no conflict of interest.

Italian ryegrass is widely cultivated for the production of forage, hay, and silage because of its high nutritional value and good palatability. Leaf spots caused by fungi pose a serious threat to forage crops. In order to expand the knowledge of fungi causing leaf spots in ryegrass ( Lolium multiflorum ) in Sichuan, Yunnan, Chongqing, and Guizhou of southwestern China, a comprehensive survey was undertaken from 2015 to 2022. The survey discovered that Epicoccum leaf spot (ELS) was a common and widespread disease, more serious at the late stage of growth (after late May). Symptomatic leaf samples collected from the four different provinces were analyzed, and a total of 202 Epicoccum isolates were obtained. Based on both multilocus phylogeny (ITS, LSU, TUB2 , and RPB2 ) and morphology, 10 Epicoccum species were finally identified, including three novel species ( E. endololii sp. nov., E. lolii sp. nov., and E. loliicola sp. nov.), six new host records ( E. draconis , E. endophyticum , E. oryzae , E. plurivorum , E. thailandicum , and E. tobaicum ), and an unknown species ( Epicoccum sp.1). Pathogenicity tests showed that E. endophyticum , E. endololii , and Epicoccum sp.1 were nonpathogenic to Italian ryegrass, which were confirmed as endophytes in this study; the other six species could infect Italian ryegrass and cause leaf lesions to different degrees, of which E. draconis was more aggressive ( P ≤ 0.05). Coupled with the isolation rates and geographical distributions of these species, it was found that E. plurivorum was the predominant pathogen in Yunnan while E. oryzae and E. tobaicum were the predominant pathogens in the other three provinces. This work provides an initial understanding of the taxonomy, virulence, and distribution of Epicoccum species associated with ELS in southwestern China and lays a solid foundation for the diagnosis in the field and scientific control of ELS on Italian ryegrass., Competing Interests: The author(s) declare no conflict of interest.

Species in the Melastomataceae (Myrtales) include trees and woody shrubs that are amongst the most common hosts of Chrysoporthe and related fungi. These fungi cause stem cankers, branch death and in extreme cases, kill their hosts. Chrysoporthe-like fungi were observed on Miconia spp. and Rhynchanthera grandiflora (Melastomataceae) plants during tree disease surveys in south-eastern Brazil including the states of Minas Gerais and Rio de Janeiro. The aims of this study were to isolate and identify the fungi utilising morphological characteristics and phylogenetic analyses. This led to the identification of a new species of Chrysoporthe described here as Chrysoporthe brasilensis sp.nov. Inoculations were conducted on R. grandiflora and M. theaezans, showing that C. brasiliensis is an aggressive pathogen. This study adds to a growing number of reports of new and pathogenic species of Chrysoporthe that potentially threaten native Myrtales globally, including important trees such as Eucalyptus, both in natural ecosystems and in planted forests., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Citrus black spot (CBS) caused by Phyllosticta citricarpa was reported for the first time in Tunisia in 2019. This was also the first reported occurrence of the disease in a Mediterranean climate. In Tunisia, CBS is mainly found in lemon (Citrus limon) orchards, and is seldom observed on sweet orange (Citrus × sinensis). This recent finding in North Africa raises questions about how the disease has been able to spread under Mediterranean climatic conditions. In this work, 216 Phyllosticta strains collected from lemon orchards in 2021, 2022 and 2023 throughout the country's main citrus-growing provinces were characterised by species morphological and molecular identification, mating type and Simple Sequence Repeats (SSR) microsatellite genotyping (MLG). P. citricarpa was the only species found to be associated with CBS in Tunisia. Although P. citricarpa is a heterothallic fungal species, potentially able to reproduce both sexually and asexually, a single mating type (MAT 1-1-1) idiomorph was found in the population. In addition, three MLGs were observed, across ten microsatellite loci, one of which was massively represented (93 %), indicating a clonal population. The clonality observed suggests a single recent introduction of the pathogen into the country. These findings support the idea that in Tunisia, P. citricarpa only reproduces asexually by pycniospores, with a relatively limited dispersal potential. This is consistent with the absence of pseudothecia on the leaf litter. These results show that CBS is able to thrive under Mediterranean conditions, even in the absence of sexual reproduction. This should be taken into consideration for CBS risk assessment and management., Competing Interests: Declaration of Competing interest None., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Bananas ( Musa spp.) are among the world's most economically important staple food crops. The most important fungal leaf diseases of Musa spp. worldwide are caused by the Sigatoka disease complex, which comprises black Sigatoka ( Pseudocercospora fijiensis ), yellow Sigatoka ( P. musae ), and Eumusae leaf spot ( P. eumusae ). Considering the rapid spreading rate of black Sigatoka in Puerto Rico since its first observation in 2004, a disease survey was conducted from 2018 to 2020 to evaluate the Sigatoka disease complex on the island. Sixty-one leaf samples showing Sigatoka-like symptoms were collected throughout the island for diagnosis by molecular approaches and fungal isolation. Molecular analysis using species-specific primers for P. fijiensis , P. musae , and P. eumusae detected the presence of P. fijiensis in 50 leaf samples. Thirty-eight fungal isolates were collected and identified by morphology and genomic sequencing from various nuclear genes. The analysis identified 24 isolates as P. fijiensis , while the rest of the isolates belonged to the genus Cladosporium spp. and Cladosporium -like spp. ( n = 5) , Neocordana musae ( n = 2), Zasmidium spp. ( n = 6), and Z. musigenum ( n = 1). The high frequency of P. fijiensis found in leaf samples and collected isolates suggests that black Sigatoka has displaced the yellow Sigatoka ( P. musae ) in Puerto Rico. Accurate identification of fungal species causing foliar diseases in Musa spp. will allow the establishment of quarantine regulations and specific management approaches in Puerto Rico., Competing Interests: The author(s) declare no conflict of interest.

Hazelnut ( Corylus avellana ), a nut crop that is rapidly expanding worldwide, is endangered by a rot. Nut rot results in hazelnut defects. A survey was conducted in northwestern Italy during 2020 and 2021 to identify the causal agents of hazelnut rots. Typical symptoms of black rot, mold, and necrotic spots were observed on hazelnuts. The prevalent fungi isolated from symptomatic hazelnut kernels were Diaporthe spp. (38%), Botryosphaeria dothidea (26%), Diplodia seriata (14%), and other fungal genera with less frequent occurrences. Among 161 isolated Diaporthe spp., 40 were selected for further analysis. Based on morphological characterization and multilocus phylogenetic analysis of the ITS, tef-1α , and tub2 , seven Diaporthe species were identified as D. eres , D. foeniculina , D. novem , D . oncostoma , D. ravennica , D. rudis , and D. sojae . D. eres was the main species isolated from hazelnut rots, in particular from moldy nuts. The pathogenicity test performed on hazelnuts 'Tonda Gentile del Piemonte' using a mycelium plug showed that all the Diaporthe isolates were pathogenic on their original host. To our knowledge, this work is the first report of D. novem , D . oncostoma , and D. ravennica on hazelnuts worldwide. D. foeniculina , D. rudis , and D. sojae were reported for the first time as agents of hazelnut rot in Italy. Future studies should focus on the comprehension of epidemiology and climatic conditions favoring the development of Diaporthe spp. on hazelnut. Prevention and control measures should target D. eres , representing the main causal agents responsible for defects and nut rot of hazelnuts in Italy., Competing Interests: The author(s) declare no conflict of interest.

Quantitative resistance is considered more durable than qualitative resistance as it does not involve major resistance genes that can be easily overcome by pathogen populations, but rather a combination of genes with a lower individual effect. This durability means that quantitative resistance could be an interesting tool for breeding crops that would not systematically require phytosanitary products. Quantitative resistance has yet to reveal all of its intricacies. Here, we delve into the case of the wheat/Septoria tritici blotch (STB) pathosystem. Using a population resulting from a cross between French cultivar Renan, generally resistant to STB, and Chinese Spring, a cultivar susceptible to the disease, we built an ultra-dense genetic map that carries 148,820 single nucleotide polymorphism (SNP) markers. Phenotyping the interaction was done with two different Zymoseptoria tritici strains with contrasted pathogenicities on Renan. A linkage analysis led to the detection of three quantitative trait loci (QTL) related to resistance in Renan. These QTL, on chromosomes 7B, 1D, and 5D, present with an interesting diversity as that on 7B was detected with both fungal strains, while those on 1D and 5D were strain-specific. The resistance on 7B was located in the region of Stb8 and the resistance on 1D colocalized with Stb19 . However, the resistance on 5D was new, so further designated Stb20q . Several wall-associated kinases (WAK), nucleotide-binding and leucine-rich repeats (NB-LRR) type, and kinase domain carrying genes were present in the QTL regions, and some of them were expressed during the infection. These results advocate for a role of Stb genes in quantitative resistance and for resistance in the wheat/STB pathosystem being as a whole quantitative and polygenic.

Pathogens cause significant challenges to global food security. On annual crops, pathogens must re-infect from environmental sources in every growing season. Fungal pathogens have evolved mixed reproductive strategies to cope with the distinct challenges of colonizing growing plants. However, how pathogen diversity evolves during growing seasons remains largely unknown. Here, we performed a deep hierarchical sampling in a single experimental wheat field infected by the major fungal pathogen Zymoseptoria tritici . We analysed whole genome sequences of 177 isolates collected from 12 distinct cultivars replicated in space at three time points of the growing season to maximize capture of genetic diversity. The field population was highly diverse with 37 SNPs per kilobase, a linkage disequilibrium decay within 200-700 bp and a high effective population size. Using experimental infections, we tested a subset of the collected isolates on the dominant cultivar planted in the field. However, we found no significant difference in virulence of isolates collected from the same cultivar compared to isolates collected on other cultivars. About 20 % of the isolate genotypes were grouped into 15 clonal groups. Pairs of clones were disproportionally found at short distances (<5 m), consistent with experimental estimates for per-generation dispersal distances performed in the same field. This confirms predominant leaf-to-leaf transmission during the growing season. Surprisingly, levels of clonality did not increase over time in the field although reproduction is thought to be exclusively asexual during the growing season. Our study shows that the pathogen establishes vast and stable gene pools in single fields. Monitoring short-term evolutionary changes in crop pathogens will inform more durable strategies to contain diseases.

Aims: Root rot caused by a group of fungi is a serious disease in mulberry. This study aims to identify and characterize Rhizopus oryzae and other fungal species associated with root rot of mulberry in India., Methods and Results: Rotted root samples were collected from the mulberry gardens from four states of Southern India. The majority of the isolates identified were R. oryzae, and others were saprophytic fungi, less abundant to occasional. Two methods of inoculations were tested to confirm the pathogenicity of the selected isolates and R. oryzae was found to be pathogenic on susceptible mulberry genotypes RC2 and SRDC-1. Multi gene phylogenetic analyses using the internal transcribed spacer region (ITS), actin (ACT) and translation elongation factor 1-α (TEF), identified the isolates as R. oryzae. Additionally, Ovatospora brasiliensis, Amesia nigricolor, Gongronella butleri, Myrmecridium schulzeri, Scedosporium boydii, Graphium euwallacea, Clonostachys rosea andTalaromyces spp. were also identified., Conclusion: This study revealed the existence of eleven species of fungi including the first report of R. oryzae and the occurrence of weak pathogens or saprophytes that are associated with the root rot of mulberry in India., Significance and Impact of the Study: This is the first report of R. oryzae causing Rhizopus rot of mulberry in India. Moreover, the occurrence of saprophytes associated with root rot of mulberry was identified. Further studies should focus more on the ability of these species to generate secondary metabolites and extracellular lytic enzymes as they are beneficial for the management of root rot disease., (© 2020 The Society for Applied Microbiology.)

The DNA barcodes were developed from ITS region for the identification of fungal plant pathogens namely, Alternaria alternata and A. tenuissima both causing leaf spots, Ascochyta rabiei causing Ascochyta blight, Fusarium oxysporum f. sp. ciceris causing wilt, Macrophomina phaseolina causing dry root rot, Rhizoctonia solani causing web blight and wet root rot, Sclerotium (Athelia) rolfsii causing collar rot, Sclerotinia sclerotiorum causing stem rot and Cercospora canescens and Pseudocercospora cruenta both causing leaf spots in pulse crops. Barcode compliance for A. alternata (DBTPQ001-18), A. tenuissima (DBTPQ002-18), A. rabiei (DBTPQ003-18), F. oxysporum f. sp. ciceris (DBTPQ004-18), M. phaseolina (DBTPQ005-18), R. solani (DBTPQ006-18), S. rolfsii (DBTPQ007-18), S. sclerotiorum (DBTPQ008-18), C. canescens (DBTPQ009-18) and P. cruenta (DBTPQ029-20) have been generated based on the Barcode of Life Data System (BOLD) system. In addition to ITS, other genomic regions were also explored and on the basis of sequence variation they were ranked as TEF-α > SSU > LSU > β-tubulin. These genes could be considered for secondary barcode and phylogenetic relatedness. ITS-based markers for the detection of A. alternata (BAA2aF and BAA2aR) and R. solani (BRS17cF and BRS17cR) were developed which provided 400 bp and 220 bp amplicons, respectively. While, for F. oxysporum f. sp. ciceris, COX1-based marker (FOCox1F and FOCox3R) was developed which amplified 150 bp. The markers proved highly specific and sensitive with detection limit of 0.0001 ng of template DNA using qPCR and simultaneously detected these three pathogens. The DNA barcodes and diagnostics developed are suitable for quick and reliable detection of these pathogens during quarantine processing and field diagnostics.

Many fungi require specific growth conditions before they can be identified. Direct environmental DNA sequencing is advantageous, although for some taxa, specific primers need to be used for successful amplification of molecular markers. The internal transcribed spacer region is the preferred DNA barcode for fungi. However, inter- and intra-specific distances in ITS sequences highly vary among some fungal groups; consequently, it is not a solely reliable tool for species delineation. Ampelomyces, mycoparasites of the fungal phytopathogen order Erysiphales, can have ITS genetic differences up to 15%; this may lead to misidentification with other closely related unknown fungi. Indeed, Ampelomyces were initially misidentified as other pycnidial mycoparasites, but subsequent research showed that they differ in pycnidia morphology and culture characteristics. We investigated whether the ITS2 nucleotide content and secondary structure was different between Ampelomyces ITS2 sequences and those unrelated to this genus. To this end, we retrieved all ITS sequences referred to as Ampelomyces from the GenBank database. This analysis revealed that fungal ITS environmental DNA sequences are still being deposited in the database under the name Ampelomyces, but they do not belong to this genus. We also detected variations in the conserved hybridization model of the ITS2 proximal 5.8S and 28S stem from two Ampelomyces strains. Moreover, we suggested for the first time that pseudogenes form in the ITS region of this mycoparasite. A phylogenetic analysis based on ITS2 sequences-structures grouped the environmental sequences of putative Ampelomyces into a different clade from the Ampelomyces-containing clades. Indeed, when conducting ITS2 analysis, resolution of genetic distances between Ampelomyces and those putative Ampelomyces improved. Each clade represented a distinct consensus ITS2 S2, which suggested that different pre-ribosomal RNA (pre-rRNA) processes occur across different lineages. This study recommends the use of ITS2 S2s as an important tool to analyse environmental sequencing and unveiling the underlying evolutionary processes., Competing Interests: No authors have competing interests.

The fungus Parastagonospora nodorum is the causal agent of septoria nodorum leaf blotch (SNB) and glume blotch which are common in many wheat growing regions in the world. The disease is complex and could be explained by multiple interactions between necrotrophic effectors secreted by the pathogen and matching susceptibility genes in wheat. An Australian P. nodorum population was clustered into five groups with contrasting properties. This study was set to identify their pathogenicity profiles using a diverse wheat panel of 134 accessions which are insensitive to SnToxA and SnTox1 in both in vitro and in vivo conditions. SNB seedling resistance/susceptibility to five representative isolates from the five clusters, responses to crude culture-filtrates (CFs) of three isolates and sensitivity to SnTox3 semi-purified effector together with 11,455 SNP markers have been used for linkage disequilibrium (LD) and association analyses. While quantitative trait loci (QTL) on 1D, 2A, 2B, 4B, 5B, 6A, 6B, 7A, 7D chromosomes were consistently detected across isolates and conditions, distinct patterns and isolate specific QTL were also observed among these isolates. In this study, SnTox3-Snn3-B1 interaction for the first time in Australia and SnTox3-Snn3-D1 interaction for the first time in bread wheat were found active using wild-type isolates. These findings could be due to new SnTox3 haplotype/isoform and exotic CIMMYT/ICARDA and Vavilov germplasm used, respectively. This study could provide useful information for dissecting novel and different SNB disease components, helping to prioritise research targets and contributing valuable information on genetic loci/markers for marker-assisted selection in SNB resistance wheat breeding programme.

Key Message: Breeding progress of resistance to fungal wheat diseases and impact of disease severity on yield reduction in long-term variety trials under natural infection were estimated by mixed linear regression models. This study aimed at quantifying breeding progress achieved in resistance breeding towards varieties with higher yield and lower susceptibility for 6 major diseases, as well as estimating decreasing yields and increasing disease susceptibility of varieties due to ageing effects during the period 1983-2019. A further aim was the prediction of disease-related yield reductions during 2005-2019 by mixed linear regression models using disease severity scores as covariates. For yield and all diseases, overall progress of the fully treated intensity (I2) was considerably higher than for the intensity without fungicides and growth regulators (I1). The disease severity level was considerably reduced during the study period for mildew (MLD), tan spot (DTR) and Septoria nodorum blotch (ear) (SNB) and to a lesser extent for brown (leaf) rust (BNR) and Septoria tritici blotch (STB), however, not for yellow/stripe rust (YLR). Ageing effects increased susceptibility of varieties strongly for BNR and MLD, but were comparatively weak for SNB and DTR. Considerable yield reductions under high disease severity were predicted for STB (-6.6%), BNR (-6.5%) and yellow rust (YLR, -5.8%), but lower reductions for the other diseases. The reduction for resistant vs. highly susceptible varieties under high severity conditions was about halved for BNR and YLR, providing evidence of resistance breeding progress. The empirical evidence on the functional relations between disease severity, variety susceptibility and yield reductions based on a large-scale multiple-disease field trial data set in German winter wheat is an important contribution to the ongoing discussion on fungicide use and its environmental impact.

PCR-based molecular approaches including RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and SRAP (sequence-related amplified polymorphism) are commonly used to analyze genetic diversity. The aims of this study are to analyze genetic diversity of M. oryzae isolates using PCR-based molecular approaches such as RAPD, ISSR, and SRAP and to develop SCAR marker linked to the pathogenicity of rice blast fungus. Twenty Magnaporthe oryzae isolates were collected mainly from the south of Vietnam and assessed for genetic variation by RAPD, ISSR, and SRAP methods. The comparison of those methods was conducted based on the number of polymorphic bands, percentage of polymorphism, PIC values, and phylogenetic analysis. Then, sequenced characterized amplified region (SCAR) markers were developed based on specific bands linked to fungal pathogenicity of rice blast fungus, M. oryzae. The results indicated that SRAP markers yielded the greatest number of polymorphic bands (174) and occupied 51.7% with polymorphism information content (PIC) value of 0.66. Additionally, the SRAP approach showed stability and high productivity compared with RAPD and ISSR. The SCAR marker developed from the SRAP method identified the presence of the avirulence AVR-pita1 gene involving fungal pathogenicity that can break down blast resistance in rice cultivars. The consistency of SCAR marker obtained in this study showed its efficiency in rapid in-field detection of fungal pathogenicity. SCAR marker developed from SRAP technique provides a useful tool for improving the efficiency of blast disease management in rice fields.

Aims: To identify Botryosphaeriaceae fungal species that are associated with stem-end rot of mango, and to study their pathogenicity on mango fruit., Methods and Results: Based on the sequences of internal transcribed spacer (ITS), TEF1-α and β-tubulin, as well as on the phylogenetic analysis of combined sequences, four species of Lasiodiplodia (L. theobromae,L. pseudotheobromae, L. iranensis, L. mahajangana) and two species of Neofusicoccum (N. ribis, N. parvum) were identified. Pseudofusicoccum violaceum, Neoscytalidium dimidiatum and three species of Botryosphaeria (B. scharifii, B. dothidea, B. ramosa) were identified based on sequences of ITS and TEF1-α. Pathogenicity test of selected isolates were tested on Chok Anan, Waterlily and Falan mango cultivars. Generally, all species were observed to be pathogenic on the three tested mango cultivars on wounded fruits, except for N. ribis and N. parvum, which were pathogenic on both wounded and unwounded fruits. However, N. ribis was only pathogenic on cultivar Falan, whereas B. ramosa were pathogenic on cultivars Waterlily and Falan., Conclusions: Eleven species of Botryosphaeriaceae were associated with mango stem-end rot in Malaysia. To the best of our knowledge, four species, namely L. mahajangana, B. ramosa, N. ribis and P. violaceum are the first recorded Botryosphaeriaceae fungi associated with stem end rot of mango., Significance and Impact of the Study: The identification of Botryosphaeriaceae fungi is important to establish suitable control measures and quarantine requirements. Many species have a wide host range, which means that there is a possibility of cross infection from other infected plants., (© 2020 The Society for Applied Microbiology.)

Among the emerging fungal diseases threatening food security, the Pseudocercospora fijiensis fungus causing black leaf streak disease of banana is one of the most marked examples of a recent worldwide pandemic on a major crop. We assessed how this pathogen spread throughout the latest invaded region, i.e. Central America and the Caribbean. We retraced its population history combining detailed monitoring information on disease outbreaks and population genetic analyses based on large-scale sampling of P. fijiensis isolates from 121 locations throughout the region. The results first suggested that sexual reproduction was not lost during the P. fijiensis expansion, even in the insular Caribbean context, and a high level of genotypic diversity was maintained in all the populations studied. The population genetic structure of P. fijiensis and historical data showed that two disease waves swept northward and southward in all banana-producing countries in the study area from an initial entry point in Honduras, probably mainly through gradual stepwise spore dispersal. Serial founder events accompanying the northern and southern waves led to the establishment of two different genetic groups. A different population structure was detected on the latest invaded islands (Martinique, Dominica and Guadeloupe), revealing multiple introductions and admixture events that may have been partly due to human activities. The results of this study highlight the need to step up surveillance to limit the spread of other known emerging diseases of banana spread mainly by humans, but also to curb gene flow between established pathogen populations which could increase their evolutionary potential., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A key factor to take actions against phytosanitary problems is the accurate and rapid detection of the causal agent. Here, we develop a molecular diagnostics system based on comparative genomics to easily identify fusariosis and specific pathogenic species as the Fusarium kuroshium, the symbiont of the ambrosia beetle Euwallaceae kuroshio Gomez and Hulcr which is responsible for Fusarium dieback disease in San Diego CA, USA. We performed a pan-genome analysis using sixty-three ascomycetes fungi species including phytopathogens and fungi associated with the ambrosia beetles. Pan-genome analysis revealed that 2,631 orthologue genes are only shared by Fusarium spp., and on average 3,941 (SD ± 1,418.6) are species-specific genes. These genes were used for PCR primer design and tested on DNA isolated from i) different strains of ascomycete species, ii) artificially infected avocado stems and iii) plant tissue of field-collected samples presumably infected. Our results let us propose a useful set of primers to either identify any species from Fusarium genus or, in a specific manner, species such as F. kuroshium, F. oxysporum, and F. graminearum. The results suggest that the molecular strategy employed in this study can be expanded to design primers against different types of pathogens responsible for provoking critical plant diseases., Competing Interests: The authors have declared that no competing interests exist.

Chickpea (Cicer arietinum L., Fabaceae) is the second most important legume after common bean (Phaseolus vulgaris L., Fabaceae) and third in production among the legumes grains worldwide. Ascochyta blight and Fusarium wilt are among the main fungal infections which cause the major losses of chickpea crop. In this work we report the phyto-pathogen controlling properties of 24 endophyte Phomopsis/Diaporthe isolates on the chickpea fungal pathogens Ascochyta rabiei, Fusarium oxysporum and Fusarium solani. The Phomopsis/Diaporthe strains were isolated amongst a total of 62 endophytic fungi from the aerial parts of the herbaceous perennial American plant Peperomia obtusifolia (Piperaceae) along with Fusarium, Septoria, Colletotrichum, Alternaria and Roussoella genera among others. Phomopsis/Diaporthe isolates were identified as Diaporthe infecunda (12 isolates), Diaporthe sackstoni (1 isolate), Diaporthe cf. brasiliensis (4 isolates) and Phomopsis cf. tuberivora (7 isolates). All the Phomopsis/Diaporthe strains antagonized A. rabiei strain AR2 with a mean of inhibition (% I) of 86.59 ± 1.49% in dual cultures. The metabolic characterization of the Phomopsis/Diaporthe strains showed groups in three clusters which were in agreement with the taxonomic identification. Bioautographic evaluation of organic extracts showed that those of D. cf. brasiliensis and D. infecunda were better as inhibitors. Strain Po 45 was one of the most active (cluster 1, 96.87% I), and its ethyl acetate extract inhibited A. rabiei growth in a bioautographic assay until at least 10 μg/mm applied showing a specific chromatographic band as the responsible of the A. rabiei inhibition.

Cheatgrass (Bromus tectorum L.) is an invasive annual grass (Poaceae) that has colonized large portions of the Intermountain West. Cheatgrass stand failures have been observed throughout the invaded region, the cause of which may be related to the presence of several species of pathogenic fungi in the soil or surface litter. In this metabarcoding study, we compared the fungal communities between sites that have and have not experienced stand failure. Samples were taken from the soil and surface litter near Winnemucca, Nevada, and in Skull Valley, Utah. Our results show distinct fungal communities associated with stand failure based on both geography and sample type. In both the Winnemucca and Skull Valley surface litter, there was an elevated abundance of the endophyte Ramimonilia apicalis in samples that had experienced a stand failure. Winnemucca surface litter stand failure samples had an increased abundance of a potential pathogen in the genus Comoclathris. Skull Valley surface litter stand failure samples had an increased abundance of an undescribed new species in the Rustroemiaceae family which is responsible for the so-called bleach blonde syndrome in cheatgrass, while the soils had an increased abundance of potential pathogens in the genera Olpidium and Monosporascus., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Phytopathogenic cercosporoid fungi have been investigated comprehensively due to their important role in causing plant diseases. A significant amount of research has been focused on the biology, morphology, systematics, and taxonomy of this group, with less of a focus on molecular or biochemical issues. Early and extensive research on these fungi focused on taxonomy and their classification based on in vivo features. Lately, investigations have mainly addressed a combination of characteristics such as morphological traits, host specificity, and molecular analyses initiated at the end of the 20th century. Some species that are important from an economic point of view have been more intensively investigated by means of genetic and biochemical methods to better understand the pathogenesis processes. Cercosporin, a photoactivated toxin playing an important role in Cercospora diseases, has been extensively studied. Understanding cercosporin toxicity in relation to reactive oxygen species (ROS) production facilitated the discovery and regulation of the cercosporin biosynthesis pathway, including the gene cluster encoding pathway enzymes. Furthermore, these fungi may be a source of other biotechnologically important compounds, e.g., industrially relevant enzymes. This paper reviews methods and important results of investigations of this group of fungi addressed at different levels over the years.

Aims: A real-time quantitative PCR (qPCR) assay was established to quantify the inoculum densities in the air and rainwater for six canker-causing pathogen groups in prune and walnut orchards in California., Methods and Results: The previously published DNA primers to target six pathogen groups including Botryosphaeria dothidea, Cytospora spp., Diplodia spp., Lasiodiplodia spp., Neofusicoccum spp. and Phomopsis spp. were used in a qPCR assay. Air samples from Burkard spore traps and rain samples from special rain collector devices were collected periodically from various prune and walnut orchards. Using the qPCR approach, we were able to quantify the concentrations of these pathogen groups in rainwater and air samples and study the dynamics of pathogen inoculum in orchards showing severe canker potential. Phomopsis spp. and Diplodia spp. were not found in all rain samples in prune orchards, although they were detected in the 2016 in the walnut orchard. The other four pathogen groups were quantified at varying concentrations in the prune and walnut orchards. Cytospora spp. in some cases showed higher concentrations in the rainwater in prune orchards., Conclusions: The rainy season during winter and early spring is a highly risky period of time for infection by the pathogens when the inoculum of these pathogens can easily spread by air and rain water, thus serving as an important inoculum source for disease initiation. The different studied pathogen groups showed different concentrations during the growing season, indicating the complexity of the components of canker-causing species in various tree crops., Significance and Impact of the Study: This study showed the applicability of the qPCR assay in the quantification of inoculum in tree orchards to help reveal the mechanisms of canker disease epidemics and to help design disease management strategies., (© 2020 The Society for Applied Microbiology.)

Diseases in plants are mostly caused by fungi. Fungal interactions with the host can be either biotrophic, necrotrophic or hemibiotrophic. Synergistic polymicrobial interactions have been recently recognized that can also attribute to the occurrence of complex plant diseases. Tea is one of the most widely consumed beverages worldwide, although tea plants are affected by many different diseases causing a significant reduction in global tea production. Blister blight is one such serious and damaging leaf disease of tea. An assessment of blister blight disease was carried out at the tea development center in Umsning, Meghalaya. A considerable number of tea varieties showed characteristic blister blight symptoms that ranged from preliminary yellow spots in the upper leaf surface, matured white sporulating blisters in the lower leaf surface, and delayed brown necrotic lesions throughout the surfaces of the leaves. A total of 42 isolates, 15 from initial, 15 from mature, and 12 from necrotic stages were isolated from the symptomatic leaf samples. Pestalotiopsis and Nigrospora were the two fungi incessantly isolated from the diseased leaves. Colony characteristics that included colony, hyphal, and spore morphologies were examined and mycelial accumulation, sporulation, and sporal germination were determined for all the isolates of Pestalotiopsis and Nigrospora. Molecular analysis based on ITS-RFLP was performed for identification and genetic variability. In vitro pathogenicity assay revealed that Pestalotiopsis spp. and Nigrospora sp. developed distinct characteristics symptoms on greenhouse acclimated TV17 tea clones. To our knowledge, this is the first report of the prevalence of tea blister blight disease in Meghalaya and it is an initial attempt to identify fungal pathogens during different stages of blister blight disease., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Plant disease is one of the most important causes of crop losses worldwide. The effective control of plant disease is related to food security. Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum leads to serious yield losses in rapeseed (Brassica napus) production. Hypovirulent strain DT-8 of S. sclerotiorum, infected with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), has the potential to control SSR. In this study, we found rapeseed bio-priming with strain DT-8 could significantly decrease the disease severity of SSR and increase yield in the field. After bio-priming, strain DT-8 could be detected on the aerial part of the rapeseed plant. By 16S rRNA gene and internal transcribed spacer (ITS) sequencing technique, the microbiome on different parts of the SSR lesion on bioprimed and non-bioprimed rapeseed stem was determined. The results indicated that SSR and bio-priming treatment could influence the structure and composition of fungal and bacterial communities. Bio-priming treatment could reduce the total abundance of possible plant pathogens and enhance the connectivity and robustness of the interaction network at the genus level. This might be one of the mechanisms that rapeseed bioprimed with strain DT-8 had excellent tolerance on SSR. It might be another possible mechanism of biocontrol and will provide a theoretical guide for agricultural practical production.

White mould or stem rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is a devastating fungal disease found in major potato cultivation areas worldwide. The aim of this study was to characterize genetic diversity in the S. sclerotiorum population from the main potato producing regions of India by means of morphological (mycelial growth, colony colour, number and distribution pattern of sclerotia) and molecular characteristics, as well as to evaluate the virulence of S. sclerotiorum isolates in potato for the first time. Among the S. sclerotiorum population analyzed, high phenotypic and genotypic diversity were observed. Using all the morphological characteristics, a dendrogram was constructed based on Gower's similarity coefficient that distributed all the isolates into three clusters at the 0.62 similarity coefficient. Carpogenic germination of apothecia revealed that larger sclerotia produced a greater number of apothecia while smaller sclerotia produced fewer apothecia. Pathogenicity test results revealed that out of 25 isolates, seven were highly aggressive, 14 were moderate and four had low aggressiveness, whilst isolates from Punjab were more pathogenic than those of Uttar Pradesh. Phylogenetic analysis of universal rice primer polymorphism showed high genetic variability within the isolates that grouped all the isolates in three evolutionary lineages in the resulting dendrogram and showed partial relationship with geographical locations of the isolates. Further, the findings suggest the occurrence of higher heterogeneity and genetic diversity among the S. sclerotiorum isolates that indicates the existence of both clonal and sexual reproduction in the pathogen population of potato producing areas in India.

Monilinia laxa is an important fungal plant pathogen causing brown rot on many stone and pome fruits worldwide. Mitochondrial genome (mitogenome) plays a critical role in evolutionary biology of the organisms. This study aimed to characterize the complete mitogenome of M. laxa by using next-generation sequencing and approaches of de novo assembly and annotation. The total length of the mitogenome of M. laxa was 178,357 bp, and its structure was circular. GC content of the mitogenome was 30.1%. Annotation of the mitogenome presented 2 ribosomal RNA (rRNA) genes, 32 transfer RNA genes (tRNA), 1 gene encoding mitochondrial ribosomal protein S3, 14 protein-coding genes and 15 open reading frame encoding hypothetical proteins. Moreover, the group I mobile introns encoding homing endonucleases including LAGLIDADG and GIY-YIG families were found both within coding regions (genic) and intergenic regions of the mitogenome, indicating an enlarged size and a dynamic structure of the mitogenome. Furthermore, a comparative mitogenomic analysis was performed between M. laxa and the three closely related fungal phytopathogen species (Botryotinia fuckeliana, Sclerotinia sclerotiorum and, S. borealis). Due to the number and distribution of introns, the large extent of structural rearrangements and diverse mitogenome sizes were detected among the species investigated. Monilinia laxa presented the highest number of homing endonucleases among the fungal species considered in the analyses. This study is the first to report a detailed annotation of the mitogenome of an isolate of M. laxa, providing a solid basis for further investigations of mitogenome variations for the other Monilinia pathogens causing brown rot disease.

Cowpea is an important pulse crop cultivated in arid and semi-arid regions of the world. During field survey, a characteristic wilt was observed in around 45 ha of cowpea fields with incidence 17-25%. Infection was seen in pre-flowering stage and infected plants showed quick wilt symptoms with tan lesions near the stem-soil interface. Fungal pathogens associated were isolated on PDA, which produced dark to grey olivaceous colonies in the centre, and aerial mycelia were appressed with floccose and white to smoke-grey. Conidia are aseptate, initially hyaline, smooth-walled, broadly ellipsoidal with rounded ends becoming dark brown. Based on these morphological features, the fungal pathogen was identified as Aplosporella sp. The ITS-rDNA region was amplified using ITS1/ITS4 primers and sequenced. The nBLAST and phylogenetic analysis confirmed the pathogen as Aplosporella hesperidica. The Koch's postulates were performed on 45-days-old cowpea plants with mycelial disc of A. hesperidica. Development of typical necrotic lesions was observed after 28 days of post-inoculation and the pathogen's identity was confirmed based on re-isolation. Efficacy of fungicides evaluated in vitro showed that the pathogen is highly sensitive to systemic fungicides rather than the contact fungicides. The cowpea production was severely affected owing to the causative agent A. hesperidica. The collar rot disease of cowpea by A. hesperidica is the first report in India. SIGNIFICANCE AND IMPACT OF THE STUDY: A new collar rot disease of cowpea recorded from India has been investigated. The necrotic lesions were enlarged and eventually quick wilt and death of the host plant was observed with incidence ranged from 17 to 25%. Associated fungal pathogen was isolated and identified as Aplosporella hesperidica based on morphology and ITS-rDNA sequence analysis. Koch's postulates were performed under greenhouse conditions and in vitro evaluation of fungicides shows that the pathogen is sensitive to systemic fungicides. This is the first report of A. hesperidica causing collar rot disease of cowpea in India., (© 2020 The Society for Applied Microbiology.)

Several Botryosphaeriaceae species are known to occur worldwide, causing dieback, canker and fruit rot on various hosts. Surveys conducted in ten commercial citrus orchards in the northern region of Algeria revealed five species of Botryosphaeriaceae belonging to three genera associated with diseased trees. Morphological and cultural characteristics as well as phylogenetic analyses of the internal transcribed spacer (ITS) region and the translation elongation factor 1-alpha (tef1-α) identified Diplodia mutila, Diplodia seriata, Dothiorella viticola, Lasiodiplodia mediterranea and a novel species which is here described as Lasiodiplodia mithidjana sp. nov.. Of these, L. mithidjana (14.1% of the samples) and L. mediterranea (13% of the samples) were the most widespread and abundant species. Pathogenicity tests revealed that L. mediterranea and D. seriata were the most aggressive species on citrus shoots. This study highlights the importance of Botryosphaeriaceae species as agents of canker and dieback of citrus trees in Algeria., Competing Interests: The authors have declared that no competing interests exist.

The genus Magnaporthiopsis of Magnaporthaceae (Magnaporthales, Sordariomycetes, Ascomycota) contains species that are predominantly necrotrophic pathogens, often producing simple hyphopodia and dark, ectotrophic runner hyphae on plant roots and stems during colonization. Fungal isolates from turfgrass roots with dark and ectotrophic runner hyphae were examined and identified based on morphological, biological, and phylogenetic analyses. Maximum likelihood and Bayesian methods were implemented to obtain phylogenetic trees for partial sequences of the 18S nuc rDNA, ITS1-5.8S-ITS2 nuc rDNA internal transcribed spacer, and 28S nuc rDNA regions, and of the minichromosome maintenance complex 7 ( MCM7 ), largest subunit of RNA polymerase II ( RPB1 ), and translation elongation factor 1-alpha ( TEF1 ) genes. Our isolates consistently formed a distinct and highly supported clade within Magnaporthiopsis . These findings were reinforced by common and distinctive biological and morphological characters. Additionally, we conducted pathogenicity evaluations and demonstrated the ability of this fungus to colonize roots of ultradwarf bermudagrass, one of its native hosts, via ectotrophic, dark runner hyphae, causing disease symptoms including root discoloration and reduced root and shoot mass. Altogether, our discoveries enabled recognition and description of a new species, Magnaporthiopsis cynodontis , which has widespread distribution in the United States.

Fungi in the Botryosphaeriaceae family cause dieback, fruit rots, and stem cankers in many tropical fruit trees. To identify which species of Botryosphaeriaceae were present in tropical fruit in Puerto Rico and the symptoms they cause in rambutan and longan, a disease survey was conducted throughout the island from 2008 to 2016. Diseased organs of rambutan, longan, mango, and tangerine were collected and 39 isolates belonging to the Botryosphaeriaceae family were isolated and identified. Phylogenetic analysis of three nuclear genes identified nine species: six Lasiodiplodia spp. and three Neofusicoccum spp. All 39 isolates were inoculated on healthy 1-year-old rambutan and longan seedlings to confirm their pathogenicity. Dieback on both rambutan and longan was observed at 14 days after inoculation (DAI). Fourteen isolates from seven Botryosphaeriaceae species ( Lasiodiplodia brasiliensis, L. hormozganensis, L. iraniensis, L. pseudotheobromae, L. theobromae, Neofusicoccum batangarum , and N. parvum ) caused dieback in rambutan. Five of these pathogenic isolates were collected from rambutan, four from longan, two from mango, and three from tangerine. Ten isolates of four Lasiodiplodia spp. caused dieback in longan: L. hormozganensis , L. iraniensis, L. pseudotheobromae and L. theobromae,. Of these, three were collected from rambutan, three from longan, one from mango, and three from tangerine. Abundant development of pycnidia on branches, called corky bark, were observed on both rambutan and longan 60 DAI. Cross-inoculations showed that pathogenicity is wide in spectrum, indicating that different planting alternatives should be considered for better crop management.

Aim: The aims of this study were to identify races and mating types of Setosphaeria turcica causing northern corn leaf blight in Heilongjiang province of China and analyse the genetic diversity of S. turcica isolates using SSR markers., Methods and Results: Based on gene-for-gene interactions, 13 races of S. turcica (races 0, 1, 2, 3, 12, 13, 23, 123, N, 1N, 12N, 3N and 23N) were isolated from infected corn plants in Heilongjiang province. Races 0 and 1 were the predominant races, and race 23N was identified for the first time in the region. Using two pairs of specific primers, three mating types, 'a', 'Aa' and 'A', were identified, with 'a' being the predominant mating type. SSR markers were used to analyse genetic diversity of 60 S. turcica isolates. Five SSR primers were polymorphic, which resulted in 45 reproducible bands with 2-15 bands for each primer. Cluster analysis separated the isolates into five groups at a similarity coefficient of 0·84. Analysis of molecular variance showed that there was significant correlation between SSR groups and mating type of the isolates. No significant correlation was found between SSR groups and physiological races or geographical location of the isolates., Conclusions: The work reported that races 0 and 1 were the predominant races, and race 23N was identified for the first time in Heilongjiang province with 'a' being the predominant mating type. There was significant correlation between SSR groups and mating type of S. turcica isolates., Significance and Impact of the Study: Our results provide information on population structure and genetic diversity of S. turcica causing Northern corn leaf blight, which will facilitate the development of effective disease management programs., (© 2019 The Society for Applied Microbiology.)

Canker and wood rot pathogens cause dieback and, in severe cases, the death of young apple trees. Recently, a higher occurrence of cankers was observed on 1-year-old apple trees in the Western Cape Province of South Africa. This study aimed to assess the phytosanitary status of nursery trees and propagation material as possible inoculum sources for canker pathogens. Thirteen 1-year-old apple orchards showing canker or dieback symptoms were sampled. Certified nursery apple trees were collected from four nurseries as well as scion and rootstock mother plant material. Isolations were made from the discoloration observed in the vascular tissue of the plant parts and from asymptomatic material. Possible canker and wood rot species were identified with PCR and sequence comparisons of the relevant gene regions and phylogenetic analyses. Similar canker and wood rot species were isolated from 1-year-old diseased apple trees, nursery apple trees, and the propagation material. Forty-five fungal species associated with canker or wood rot symptoms were identified. The top five most abundant fungal species found causing disease on commercial 1-year-old trees were also found in high numbers causing latent infection in certified apple nursery trees. These species were Didymosphaeria rubi-ulmifolii sensu lato, Diplodia seriata , Schizophyllum commune , Didymella pomorum , and Coniochaeta fasciculata , with D. rubi-ulmifolii sensu lato being the dominant species in both sampling materials. In all, 65% of certified nursery apple trees, 5% of scion shoots used for budding, and 21% of rooted rootstock cuttings from layer blocks had latent infections of canker and wood rot pathogens. Pathogenicity trials were conducted with isolates of 39 species, inoculated onto 2-year-old branches of 14-year-old Golden Delicious trees. All species caused lesions that were significantly longer than the control. This study confirmed the presence of canker and wood rot pathogens in apple propagation material as well as certified nursery apple trees, which will aid the improvement of management practices in nurseries.

Leaf blight and fruit rot disease caused by Phomopsis vexans is a devastating disease of brinjal. The detection of P. vexans in plant parts and seeds of brinjal can be complicated, mainly when the inoculum is present at low levels and/or overgrown by fast-growing saprophytic fungi or other seed-borne fungi. A PCR-based diagnostic method was developed with specific primers designed based on sequence data of a region consisting of the 5·8S RNA gene and internal transcribed spacers, ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of P. vexans. The efficiency and specificity of primer pairs PvexF/PvexR designed were established by PCR analysis of DNA from P. vexans strains isolated from India and fungal isolates of other genera. A single amplification product of 323-bp was detected from DNA of P. vexans isolates. No cross-reaction was observed with any of the other isolates tested. The specific primers designed and employed in PCR detected P. vexans up to 10 pg from DNA isolated from pure culture. This is the first report on the development of species-specific PCR assay for identification and detection of P. vexans. Thus, PCR-based assay developed is very specific, rapid, confirmatory and sensitive tool for the detection of pathogen P. vexans at early stages. SIGNIFICANCE AND IMPACT OF THE STUDY: Phomopsis vexans is an important seed-borne pathogenic fungus responsible for leaf blight and fruit rot in brinjal. Current detection methods, based on culture and morphological identification is time consuming, laborious and are not always reliable. A PCR-based diagnostic method was developed with species-specific primers designed based on sequence data of a region consisting of the 5·8S RNA gene and internal transcribed spacers, ITS 1 and ITS 2 of nuclear ribosomal RNA gene (rDNA) repeats of P. vexans., (© 2019 The Society for Applied Microbiology.)

Species of Septoria are commonly associated with leaf spot diseases of a broad range of plant hosts worldwide. During our investigation of fungi associated with leaf spot diseases in northern and northwestern Iran, several Septoria isolates were recovered from symptomatic leaves on different herbaceous and woody plants in the Asteraceae, Betulaceae, and Salicaceae families. These isolates were studied by applying a polyphasic approach including morphological and cultural data and a multigene phylogeny using a combined data set of partial sequences of the 28S nuc rRNA gene (large subunit [28S]), internal transcribed spacer regions and intervening 5.8S nuc rRNA gene (ITS) of the nuc rDNA operon, actin ( actA ), translation elongation factor 1-α ( tef1 ), calmodulin ( cmdA ), β-tubulin ( tub2 ), and DNA-directed RNA polymerase II second largest subunit ( rpb2 ). Four novel species are proposed, namely, Septoria eclipticola on Eclipta prostrata, Septoria firouraghina on Cirsium arvense, Septoria guilanensis on Populus deltoides , and Septoria taleshana on Alnus subcordata . All species are illustrated, and their morphology and phylogenetic relationships with other Septoria species are discussed.

Septoria tritici blotch (STB) caused by the heterothallic ascomycete Zymoseptoria tritici is currently one of the most devastating diseases of wheat worldwide. The extent of sexual reproduction of this pathogen is well documented on bread wheat, but not on durum wheat. The objective of the present study was to quantify the occurrence of Z. tritici sexual reproduction on durum wheat in the Tunisian environment. The assessment was undertaken using a triple approach combining fruiting body assessment, ascospore trapping and population genetic analyses. The results highlighted the formation of pseudothecia on leaves and stubble from the autumn until the end of the growing season. Likewise, qPCR monitoring highlighted a constant release of Z. tritici airborne inoculum during the wheat-growing season, with a peak of production at the end of the season. Genetic investigations using microsatellites revealed high levels of gene and genotypic diversities, an equal distribution of mating types, and a lack of genetic clustering within and between growing seasons. Taken together, these findings indicate that Z. tritici undergoes sexual reproduction on durum wheat in Tunisia at least to the same extent than on bread wheat in Western Europe, and that the dry and warm climate does not affect the mating process of the fungus. Frequent occurrence of sexual reproduction is a valuable knowledge to take into account in STB control strategies on durum wheat., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Several Pestalotiopsis -like species cause gray blight disease in tea plants, resulting in severe tea production losses. However, systematic and comprehensive research on the diversity, geographical distribution, and pathogenicity of pathogenic species associated with tea plants in China is limited. In this study, 168 Pestalotiopsis -like isolates were obtained from diseased tea plant leaves from 13 primary tea-producing provinces and cities in China. Based on a multilocus (internal transcribed spacer, translation elongation factor 1-α, and β-tubulin gene region) phylogenetic analysis coupled with an assessment of conidial characteristics, 20 Neopestalotiopsis unclassified isolates, seven Pestalotiopsis species, including two novel ( Pestalotiopsis menhaiensis and Pestalotiopsis sichuanensis ), four known ( Pestalotiopsis camelliae , Pestalotiopsis chamaeropis , Pestalotiopsis kenyana , and Pestalotiopsis rhodomyrtus ) and one indistinguishable species, and three Pseudopestalotiopsis species, including two known ( Pseudopestalotiopsis camelliae-sinensis and Pseudopestalotiopsis chinensis ) and one indistinguishable species, were identified. This study is the first to evaluate Pestalotiopsis chamaeropis on tea plants in China. The geographical distribution and pathogenicity tests showed Pseudopestalotiopsis camelliae-sinensis to be the dominant cause of gray blight of tea plants in China. In vitro antifungal assays demonstrated that theobromine not only derepressed mycelial growth of the 29 representative isolates but also increased their growth. Correlation analysis revealed a linear positive relationship between the mycelial growth rate and pathogenicity ( P = 0.0148).

In field and laboratory experiments during 2014-2017, we investigated the influence of lower and higher cultivation intensity of wheat and ecological factors (weather-temperature and rainfalls, year) on the occurrence of phytopathogenic fungi on the leaves of winter wheat. The prevailing fungi in those years were Mycosphaerella graminicola (Fuckel) J. Schrott and Pyrenophora tritici-repentis (Died.) Drechsler. Using cluster analysis, we statistically evaluated interrelationships of known factors on the abundance of the fungi on leaf surfaces. Our results showed strongest correlation with Mycosphaerella graminicola and Pyrenophora tritici-repentis abundance to be with lower cultivation intensity and year done by the temperature and the rainfalls. The two pathogens-Puccinia tritici Oerst and Hymenula cerealis Ellis & Everh. occurred only very sparsely in some years and had little positive or negative correlation with named factors. The semi-early and semi-late winter wheat varieties Matchball, Annie, Fakir, and Tobak were used for our experiments. Higher cultivation intensity had protective effect against leaf phytopathogenic fungi.

The internal transcribed spacer (ITS) DNA marker is routinely used for fungal identification but gives a clear result for only three out of four powdery mildew samples. A search for new markers indicates that some genes offer enhanced identification in comparison with ITS. Others fail due to amplification and sequencing difficulties and lack of informative variability. Powdery mildews (Ascomycota, Erysiphales) are biotrophic, fungal plant pathogens that commonly occur worldwide on a wide range of host plants. They are unsightly and greatly reduce the vigor of their hosts and have major impacts on crop and other cultivated plants. Species within this order are straightforward to spot, but difficult to identify. A citizen science scheme was run in 2013-2016 in the UK to gather a wide array of samples on which identification methods could be developed. Current techniques for identification and phylogenetic reconstruction show scope for improvement. In this paper, we review genes used in other fungal groups for discrimination at species level. Working protocols for amplification and sequencing of seven genes ( actin, β-tubulin, calmodulin , Chs , elongation factor 1-α [ EF1-α ], Mcm7 , and Tsr1 ) are developed with varying success; Mcm7 proves to be the most useful at differentiation between closely related, phylogenetically young powdery mildew species for phylogenetic reconstruction when used separately and in tandem with ITS. We therefore propose this as the most appropriate candidate gene to be used commonly in powdery mildew diagnostics alongside the ITS; furthermore, this could be transferred to similarly troublesome fungal clades.

Blackleg, caused by the fungus Leptosphaeria maculans , is a widespread disease of winter canola ( Brassica napus ) in Oklahoma and Kansas. Deployment of genetic resistance is the primary strategy for managing blackleg. Resistance genes ( Rlm ) in canola interact with avirulence genes in the fungus ( AvrLm ) in a gene-for-gene manner. Little is known about the diversity and frequency of avirulence genes and the race structure in the region. Isolates of Leptosphaeria spp. were collected from diseased leaves in nine counties in Oklahoma and one county in Kansas from 2009 to 2013. Based on pathogenicity and PCR amplification of mating type and species-specific internal transcribed spacer loci, most isolates ( n = 90) were L. maculans. The presence of avirulence genes was evaluated using phenotypic interactions on cotyledons of differential cultivars with Rlm1 , Rlm2 , Rlm3 , and Rlm4 and amplification of AvrLm1 , AvrLm4-7 , and AvrLm6 by PCR. The avirulence alleles AvrLm6 and AvrLm7 were present in the entire L. maculans population. AvrLm1 was found in 34% of the population, AvrLm2 in 4%, and AvrLm4 in only 1%. A total of five races, defined as combinations of avirulence alleles, were identified that included AvrLm1-2-6-7 , AvrLm2-6-7 , AvrLm4-6-7 , AvrLm1-6-7 , and AvrLm6-7 . Races virulent on the most Rlm genes, AvrLm1-6-7 at 32% and AvrLm6-7 at 62%, were predominant. Defining the avirulence allele frequency and race structure of L. maculans should be useful for the identification and development of resistant cultivars and hybrids for blackleg management in the region. The results suggest that Rlm6 and Rlm7 would be effective, although their deployment should be integrated with quantitative resistance and cultural practices, such as crop rotation, that limit selection pressure on Rlm genes.

Synchytrium endobioticum is an obligate biotrophic soilborne Chytridiomycota (chytrid) species that causes potato wart disease, and represents the most basal lineage among the fungal plant pathogens. We have chosen a functional genomics approach exploiting knowledge acquired from other fungal taxa and compared this to several saprobic and pathogenic chytrid species. Observations linked to obligate biotrophy, genome plasticity and pathogenicity are reported. Essential purine pathway genes were found uniquely absent in S. endobioticum, suggesting that it relies on scavenging guanine from its host for survival. The small gene-dense and intron-rich chytrid genomes were not protected for genome duplications by repeat-induced point mutation. Both pathogenic chytrids Batrachochytrium dendrobatidis and S. endobioticum contained the largest amounts of repeats, and we identified S. endobioticum specific candidate effectors that are associated with repeat-rich regions. These candidate effectors share a highly conserved motif, and show isolate specific duplications. A reduced set of cell wall degrading enzymes, and LysM protein expansions were found in S. endobioticum, which may prevent triggering plant defense responses. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, reflects characteristic biological differences between the phyla, and shows commonalities in genomic features among pathogenic fungi.

Persian lime ( Citrus latifolia Tan.) is an important and widely cultivated fruit crop in several regions of Mexico. In recent years, severe symptoms of gummosis, stem cankers, and dieback were detected in the Persian lime-producing region in the states of Veracruz and Puebla, Mexico. The aims of this study were to identify the species of Lasiodiplodia associated with these symptoms, determine the distribution of these species, and test their pathogenicity and virulence on Persian lime plants. In 2015, symptomatic samples were collected from 12 commercial Persian lime orchards, and 60 Lasiodiplodia isolates were obtained. Fungal identification of 32 representative isolates was performed using a phylogenetic analysis based on DNA sequence data of the internal transcribed spacer region and part of the translation elongation factor 1-α and β-tubulin genes. Sequence analyses were carried out using the Maximum Likelihood and Bayesian Inference methods. Six Lasiodiplodia species were identified as Lasiodiplodia pseudotheobromae , Lasiodiplodia theobromae , Lasiodiplodia brasiliense , Lasiodiplodia subglobosa , Lasiodiplodia citricola , and Lasiodiplodia iraniensis . All Lasiodiplodia species of this study are reported for the first time in association with Persian lime in Mexico and worldwide. L. pseudotheobromae (46.9% of isolates) was the most frequently isolated species followed by L. theobromae (28.1%) and L. brasiliense (12.5%). Pathogenicity on Persian lime young plants using a mycelial plug inoculation method showed that all identified Lasiodiplodia species were able to cause necrotic lesions and gummosis, but L. subglobosa , L. iraniensis , and L. pseudotheobromae were the most virulent.

Didymella pisi is the predominant causal pathogen of ascochyta blight of dry pea causing yield losses in Montana, where 415 000 acres were planted to dry pea in 2018. Thirty-three microsatellite markers were developed for dry pea pathogenic fungus, Didymella pisi, these markers were used to analyze genetic diversity and population structure of 205 isolates from four different geographical regions of Montana. These loci produced a total of 216 alleles with an average of 1.63 alleles per microsatellite marker. The polymorphic information content values ranged from 0.020 to 0.990 with an average of 0.323. The average observed heterozygosity across all loci varied from 0.000 to 0.018. The gene diversity among the loci ranged from 0.003 to 0.461. Unweighted Neighbor-joining and population structure analysis grouped these 205 isolates into two major sub-groups. The clusters did not match the geographic origin of the isolates. Analysis of molecular variance showed 85 % of the total variation within populations and only 15 % among populations. There was moderate genetic variation in the total populations (PhiPT = 0.153). Information obtained from this study could be useful as a base to design strategies for improved management such as breeding for resistance to ascochyta blight of dry pea in Montana., (Published by Elsevier Ltd.)

Persimmon (Diospyros kaki) fruit production is severely affected by circular leaf spot worldwide. Mycosphaerella nawae causes circular leaf spot of persimmon (CLSP) and can result in leaf spot, defoliation, early fruit maturation, and subsequent softening and abscission. The morphology and phylogenetic position of M. nawae within the family Mycosphaerellaceae is, therefore, of utmost importance given its impact on persimmon production. Based on previous morphological and molecular studies, the phylogenetic position of the anamorphic genera associated with M. nawae remain in confusion. In the present study, 15 isolates of M. nawae were collected from the tissue of living leaves exhibiting leaf spot symptoms. A subsample of three isolates was characterized phylogenetically and morphologically. Isolates were compared based on DNA sequence data for the internal transcribed spacer region (ITS1-5.8S ITS2), part of the 28S nrDNA including domains D1-D3 (LSU), actin (Act), translation elongation factor 1-alpha (EF-1α), and RNA polymerase II second largest subunit (rpb2). The anamorph and teleomorph structures, ascospore germination patterns, as well as host specificity were used to describe the isolates. The phylogenetic and morphological analyses revealed that M. nawae requires a new holomorphic genus within Mycosphaerellaceae, described herein as Plurivorosphaerella gen. nov. A host specificity test revealed that Plurivorosphaerella nawae comb. nov. (M. nawae) can superficially colonize, but not infect, apple, peach, cherry, and plum.

Background: DNA metabarcoding, commonly used in exploratory microbial ecology studies, is a promising method for the simultaneous in planta-detection of multiple pathogens associated with disease complexes, such as the grapevine trunk diseases. Profiling of pathogen communities associated with grapevine trunk diseases is particularly challenging, due to the presence within an individual wood lesion of multiple co-infecting trunk pathogens and other wood-colonizing fungi, which span a broad range of taxa in the fungal kingdom. As such, we designed metabarcoding primers, using as template the ribosomal internal transcribed spacer of grapevine trunk-associated ascomycete fungi (GTAA) and compared them to two universal primer widely used in microbial ecology., Results: We first performed in silico simulations and then tested the primers by high-throughput amplicon sequencing of (i) multiple combinations of mock communities, (ii) time-course experiments with controlled inoculations, and (iii) diseased field samples from vineyards under natural levels of infection. All analyses showed that GTAA had greater affinity and sensitivity, compared to those of the universal primers. Importantly, with GTAA, profiling of mock communities and comparisons with shotgun-sequencing metagenomics of field samples gave an accurate representation of genera of important trunk pathogens, namely Phaeomoniella, Phaeoacremonium, and Eutypa, the abundances of which were over- or under-estimated with universal primers., Conclusions: Overall, our findings not only demonstrate that DNA metabarcoding gives qualitatively and quantitatively accurate results when applied to grapevine trunk diseases, but also that primer customization and testing are crucial to ensure the validity of DNA metabarcoding results.