Fungal microbiota in seeds,seedlings and mature plants of raspberry (Rubus ideaus L.)

Presently, there is an intensive search for fungal endophytes to be used in agriculture for the protection and condition improvement of plants and in medicine. We screened for the presence of endophytes in raspberry, which occurs naturally in the Bia?owie?a Forest. The fungal isolates representative of each morphotype were analysed using the molecular markers ITS1 and ITS2. In total, we found 34 taxa of endophytic fungi. The majority were potential pathogens. As many as 27 taxa were found in the leaves of mature plants. No fungi could be isolated from the surface sterilized seeds obtained from these plants. Seedlings were grown from the seeds deposited in the soil seed bank in the Bia?owie?a Geobotanical Station of the University of Warsaw in Bia?owie?a. 8 taxa of endophytic fungi were found in seedlings. It could be due to a possibility of seed infection with these endophytes in soil conditions.

     

Significant in vitro antagonism of the laurel wilt pathogen by endophytic fungi from the xylem of avocado does not predict their ability to control the disease

The ascomycete Raffaelea lauricola causes laurel wilt, a lethal vascular disease of avocado, Persea americana, and other members of the Lauraceae plant family. Few effective control measures for laurel wilt exist and new measures are needed. In this study, biological control of the disease with endophytic fungi from avocado was examined. Thirty‐two endophytes (24 operational taxonomic units or OTUs) isolated from the xylem of healthy trees (the infection court of R. lauricola) were evaluated against R. lauricola with in vitro dual‐culture assays. Nine OTUs that showed strong in vitro antagonism of the pathogen were tested in planta against laurel wilt. In three greenhouse experiments, grafted avocado plants of Simmonds or Russell cultivars, which are both susceptible to laurel wilt, were inoculated with endophytes and, after 10–16 days, inoculated with the same isolate of R. lauricola that was used in the in vitro assays. Within 14 days of inoculation with R. lauricola, laurel wilt developed in plants that were not treated with endophytes (positive controls) but also developed in endophyte‐treated plants to the extent observed in the positive controls (P = 0.05). The pathogen colonized plants rapidly and systemically, but endophytes generally did not colonize xylem more than 2 cm above the point at which plants were inoculated. Although the tested endophytes strongly antagonized the pathogen in vitro, this did not translate to an ability to reduce development of laurel wilt. The management of laurel wilt and other plant diseases with fungal endophytes is discussed.     

Fusarium species associated with stalk rot and head blight of grain sorghum in Queensland and New South Wales,Australia

Historical records report Fusarium moniliforme sensu lato as the pathogen responsible for Fusarium diseases of sorghum; however, recent phylogenetic analysis has separated this complex into more than 25 species. During this study, surveys were undertaken in three major sorghum‐producing regions in eastern Australia to assess the diversity and frequency of Fusarium species associated with stalk rot‐ and head blight‐infected plants. A total of 523 isolates were collected from northern New South Wales, southern Queensland and central Queensland. Nine Fusarium species were isolated from diseased plants. Pathogenicity tests confirmed F. andiyazi and F. thapsinum were the dominant stalk rot pathogens, whilst F. thapsinum and species within the F. incarnatum–F. equiseti species complex were most frequently associated with head blight.     

Identification of Trichoderma SKT-1, a biological control agent against seedborne pathogens of rice

Trichoderma SKT-1 was previously reported as a powerful biological control agent against seedborne pathogens of rice, but the taxonomic disposition of the fungal isolate was not clear. Trichoderma SKT-1 produced irregular pyramidal warts on conidia and had an optimum growth temperature of 30°C. Morphological characteristics and colony growth were identical to those of known species of Trichoderma, including the newly recognized species T. asperellum. The 5.8S rDNA with the internal transcribed spacer (ITS) region (ca. 514 bp) of the fungus was compared with those of known species to determine the phylogenetic placement of the fungus. The length and sequence of the regions from Trichoderma SKT-1 were completely identical to those of an isolate of T. asperellum NRRL 5242 (AJ230669). On the basis of these results, we concluded that Trichoderma SKT-1 was T. asperellum.     

Endophytic fungi in the invasive weed Impatiens glandulifera: a barrier to classical biological control?

The rust fungus, Puccinia komarovii var. glanduliferae, has been introduced into the UK for biological control of the invasive weed, Impatiens glandulifera (Himalayan balsam). However, establishment of the pathogen has differed across the country, which may be partly explained by variation in plant genotype. The aim of this study was to examine whether there is a further layer of phenotypic resistance, provided by indigenous foliar endophytic fungi. Culturable endophytes were isolated from a number of different balsam populations, and the commonest species were inoculated into ‘clean’ balsam plants, to test their interactions with the rust. We found that endophyte communities within balsam are low in diversity and become more dissimilar with increasing distance between populations. Three endophytes (Colletotrichum acutatum, Alternaria alternata and Cladosporium oxysporum) were common and appeared to be antagonistic to the rust, reducing pustule number and mitigating the effect of the pathogen on plant biomass. I. glandulifera thus partially conforms to the endophyte-enemy release hypothesis, in that as an introduced species, it has an impoverished endophyte complement, acquired from the local environment. However, these endophytes represent a potential barrier to effective biological control and future weed control strategies need to find strains of rust that can overcome plant genetic resistance and the overlaying phenotypic resistance, conferred by endophytes. Future classical biological control programmes of weeds must therefore take into account the fungal bodyguards that invasive species may acquire in their introduced ranges.     

Characteristics and distribution of Colletotrichum species in coffee plantations in Hainan,China

Anthracnose caused by Colletotrichum species is a serious disease on a range of economically important hosts. To determine the Colletotrichum species in coffee plantations in Hainan, China, 55 isolates were obtained from Coffea arabica (arabica) and C. canephora var. robusta (robusta) in five counties. Initially, partial sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to measure fungal genetic diversity. Then a subset of 23 isolates was selected to represent the range of genetic diversity, varieties and geographic origin for further multilocus phylogenetic analyses. These isolates belong to eight known Colletotrichum species from three Colletotrichum species complexes, including gloeosporioides (C. endophytica, C. fructicola, C. ledongense, C. siamense and C. tropicale), boninense (C. karstii), gigasporum (C. gigasporum), and one singleton species (C. brevisporum). Of these, C. siamense was isolated in all sampled counties and C. fructicola was identified in three counties. The other six species were isolated only in one or two counties. Only C. siamense and C. fructicola were isolated from arabica, whereas all eight species were isolated from robusta. Occurrence of C. brevisporum, C. endophytica, C. ledongense and C. tropicale in coffee has not been reported previously. Pathogenicity tests showed that all eight species were pathogenic to coffee leaves and fruit. In vitro tests showed that Colletotrichum isolates from coffee in Hainan were most sensitive to prochloraz, less sensitive to carbendazim, propiconazole and difenoconazole, and least sensitive to myclobutanil.     

Cold plasma as a novel treatment to reduce the in vitro growth and germination of Colletotrichum species

This study investigated the use of cold plasma to reduce the in vitro growth of two postharvest fungal plant pathogens, Colletotrichum alienum and C. fioriniae, isolated from avocados. Cold plasma (CP) was used to treat pure cultures and conidial suspensions of both pathogens, for 180 or 360 s, in either open or sealed environments from varying distances. In an open environment, the 360 s treatment at a distance of 5 cm reduced the colony growth of freshly inoculated cultures to less than 2 mm/day compared to the control of more than 8 mm/day, and treatment of conidial suspensions resulted in almost 100% reduction of conidial germination. In the same environment, the 180 s CP treatment did not significantly reduce the colony growth of fresh or actively growing cultures, but did suppress the germination of conidia by up to 80%. In a sealed environment, the 360 s CP treatment also effectively reduced the growth of freshly inoculated cultures, with no growth for some isolates. Production of reactive oxygen and nitrogen species was observed during treatment, and these may have contributed to the reduction in growth and germination. These results demonstrate the potential of CP for the control of two Colletotrichum species.     

Performance of three endophytic actinomycetes in relation to plant growth promotion and biological control of <Emphasis Type="Italic">Pythium aphanidermatum</Emphasis>, a pathogen of cucumber under commercial field production conditions in the United Arab Emirates

In the current study, the performance of three endophytic actinomycetes identified as Actinoplanes campanulatus, Micromonospora chalcea and Streptomyces spiralis previously shown to reduce seedling damping-off, and root and crown rots of mature cucumber (Cucumis sativus) caused by Pythium aphanidermatum in pots under greenhouse conditions were further evaluated to determine their potential as biological control agents and as plant growth promoters in the field under the conditions of commercial production of cucumbers in the United Arab Emirates (UAE). When applied individually or in combination to cucumber seedlings, the three isolates significantly promoted plant growth and yield and reduced seedling damping-off and root and crown rots of mature cucumber plants. Individually the performance level of S. spiralis was relatively the best followed by A. campanulatus and then by M. chalcea. The three isolates (which were not inhibitory to each other) performed better, both as biological control agents as well as plant growth promoters, when applied together than when they were inoculated individually. The ability of these three isolates to colonize the internal tissues of roots, stems and leaves under field conditions, and to persist up to 8 weeks after seedling inoculation, showed that they can easily adapt to an endophytic habit systemically within healthy cucumber plants. As the three endophytic actinomycete isolates also colonized the rhizosphere and showed outstanding rhizosphere competency it is clear that they are facultative and not obligate endophytes. The success with the three inoculants indicated that they could well be used in place of the fungicide metalaxyl which is currently recommended for the management of Pythium diseases in the UAE. This is the first successful field use of endophytic actinomycetes as promising plant growth promoters and biological control agents against Pythium diseases of cucumber.     

Influence of native endophytic bacteria on the growth and bacterial crown rot tolerance of papaya (Carica papaya)

The native plant microbiome is composed of diverse microbial communities that influence overall plant health, with some species known to promote plant growth and pathogen resistance. Here, we show the antibacterial and growth promoting activities of autoclaved culture metabolites (ACM) from native endophytic bacteria (NEB). These NEB were isolated from a papaya cultivar (var. Cariflora) that is tolerant to bacterial crown rot (BCR) caused by Erwinia mallotivora. In this cultivar, bacterial colonization in tissues recovering from the disease was observed before onset of tissue regeneration or ‘regrowth’. We further isolated and characterized these bacteria and were able to identify two culturable stem NEB related to plant endophytic genera Kosakonia sp. (ex. Enterobacter sp., isolate EBW), and to Sphingomonas sp. (isolate EBY). We also identified root NEB under genus Bacillus (isolates BN, BS, and BT). Inhibition assays indicated that ACM from these NEB promptly (within 18-30 h) and efficiently inhibited (60–65% reduction) E. mallotivora proliferation in vitro. When surface-sterilized papaya seeds were soaked in ACM from isolates EBY and EBW, germination was variably retarded (20–60% reduction) depending on plant genotype, but plant biomass accumulation was significantly stimulated, at around two-fold increase. Moreover, greenhouse experiments show that ACM from all isolates, especially isolate EBW, significantly reduced BCR incidence and severity in a susceptible genotype (var. Solo), at around two-fold. In general, our observations of pathogen antagonism and plant growth promotion leading to disease reduction, suggested the influence of native endophytic bacteria to increased fitness in plants, and tolerance against the re-emerging crown rot disease of papaya.

     

Multilocus identification and thiophanate-methyl sensitivity of Colletotrichum gloeosporioides species complex associated with fruit with symptoms and symptomless leaves of mango

Colletotrichum spp. are known causal agents of anthracnose in a broad host range, causing severe losses. Currently, the most effective way to reduce disease is by fungicide application, which could give rise to resistant populations. This study aimed to determine the Colletotrichum species present in conventional and organic mango orchards and to evaluate their pathogenicity and sensitivity to the benzimidazole fungicide thiophanate-methyl. Seventy-one isolates from fruit with symptoms and symptomless leaves were obtained. From these, 20 representative morphotypes were analysed based on glyceraldehyde-3-phosphate dehydrogenase partial gene sequencing. A subset of 10 isolates based on different species, isolation source, and fungicide sensitivity was used for morphological and multilocus phylogenetic analysis. Colletotrichum queenslandicum was only identified in conventional production systems, C. chrysophilum only in organic systems, and C. asianum and C. siamense in both. Pathogenicity tests showed all species were pathogenic, and only C. asianum caused symptoms via both unwounded and wounded inoculation methods. Overall, 25.3% of isolates (n = 18) that belong to C. siamense, isolated from a conventional orchard, grew on thiophanate-methyl amended media at 1,000 µg/ml, suggesting high resistance. Resistance was not correlated with any common point mutations at positions 198 and 200 of the β-tubulin 2 protein, as commonly found in other fungal pathogens resistant to benzimidazole. The 74.7% remaining isolates (n = 53) belonging to the other species were sensitive, reaching 100% inhibition at <10 µg/ml. Even with benzimidazole application, anthracnose symptoms persist due to the emergence of pathogenic Colletotrichum subpopulations that are resistant to thiophanate-methyl.     

Identification of fungal pathogens and analysis of genetic diversity of Fusarium tricinctum causing root rots of alfalfa in north-east China

Alfalfa root rot is a devastating disease complex found worldwide. Population structure and genetic diversity of fungal pathogens causing alfalfa root rot in north-east China are not well understood. In this study, 480 fungal isolates were collected from six major alfalfa-growing regions in Heilongjiang province, China. They were identified as Fusarium tricinctum, F. oxysporum, F. acuminatum, F. solani, F. equiseti, Phoma medicaginis, Plectosphaerella cucumerina, Alternaria alternata, and Chaetomium globosum and caused root rot on alfalfa in greenhouse studies. F. tricinctum was the predominant species among the isolates, and P. medicaginis and C. globosum had not previously been reported causing alfalfa root rot in north-east China. Of the 73 F. tricinctum isolates identified, the majority were moderately or highly aggressive on alfalfa. No isolate of F. tricinctum was sensitive to carbendazim (1 and 10 μg/ml), indicating that, although commonly used, it is not suitable for management of the disease in this area. F. tricinctum isolates were analysed using AFLP markers and divided into eight genetic groups with 28 pairs of primers. Analysis of molecular variance indicated significant correlation between genetic groups of F. tricinctum isolates and their geographical locations or aggressiveness. Pairwise comparison and STRUCTURE analysis also indicated that geographical locations and aggressiveness of isolates had a significant effect on population differentiation. This study provides insight into the genetic diversity and reproductive biology of F. tricinctum, enhances understanding of the population diversity of alfalfa root rot pathogens in north-east China, and facilitates development of effective strategies for managing this destructive disease complex.     

Most Colletotrichum species associated with tree tomato (Solanum betaceum) and mango (Mangifera indica) crops are not host‐specific

An important constraint for crop production in Colombia is the high incidence of anthracnose caused by Colletotrichum species. Although several studies have focused on these fungi, the relationship between the different fungal species within the genus and their hosts and whether they display any host preference or host specificity has yet to be examined. In Colombia, diseases caused by Colletotrichum species are particularly severe in mango (Mangifera indica) and tree tomato (Solanum betaceum) crops. In a previous investigation, the Colletotrichum phylogenetic species attacking these crops were identified. The present study aimed to determine whether isolates collected from tree tomato and mango showed host preference or host specificity by assessing aggressiveness, spore density, latent period, and fitness of each strain on the two hosts. In the departments of Cundinamarca and Tolima, Colombia, isolates were collected from plants that presented typical anthracnose symptoms and were identified as C. acutatum, C. asianum, C. boninense, C. gloeosporioides, C. tamarilloi and C. theobromicola. Inoculation of conidia of each isolate onto both hosts showed isolates had no host preference and only the C. gloeosporioides isolate showed host specificity. However, in general, isolates produced a higher spore density when inoculated on the alternate host, which may indicate a difference in the degree of adaptation to each host. Statistical analyses of the assessed parameter values revealed that isolates use different infection strategies when infecting each host. In light of these results, the implications of using quantitative estimations of fitness when studying fungal pathogens are discussed.     

Fungal endophyte protects Atractylodes lancea from root rot caused by Fusarium oxysporum

Endophytic fungi, which stimulate a variety of defence reactions in host plants without causing visible disease symptoms, have been isolated from almost every plant. However, beneficial interactions between fungal endophytes and pathogens from the same habitat remain largely unknown. An inoculation of Atractylodes lancea plantlets with Gilmaniella sp. AL12 (AL12) prior to infection with Fusarium oxysporum prevented the necrotization of root tissues and plant growth retardation commonly associated with fusarium root rot. Quantification of F. oxysporum infections using real‐time PCR revealed a correlation between root rot symptoms and the relative amount of fungal DNA. Pretreatment with AL12 reduced the accumulation of reactive oxygen species stimulated by F. oxysporum. An in vitro analysis of their interactions under axenic culture conditions showed AL12 could inhibit F. oxysporum growth. Additionally, F. oxysporum infections were shown to decrease salicylic acid (SA) production compared with control plantlets. SA biosynthesis inhibitors, 2‐aminoindan‐2‐phosphonic acid and paclobutrazol, abolished the inhibition of F. oxysporum growth in A. lancea even after inoculation with AL12. The results indicated that the fungal endophyte protected A. lancea not only by direct antibiosis, but also by reversing the F. oxysporum‐mediated suppression of SA production.     

Development of three fusarium crown rot causal agents and systemic translocation of deoxynivalenol following stem base infection of soft wheat

Fusarium pseudograminearum, F. culmorum and F. graminearum are the most important fusarium crown rot (FCR) causal agents. They have the common ability to biosynthesize deoxynivalenol (DON). To elucidate the behaviour of each of the three species, a comparative study was carried out to investigate symptom progression, fungal systemic growth and translocation of DON following stem base inoculation of soft wheat. FCR symptoms were mainly localized in the inoculated area, which extended up to the second node for all inoculated species. Only the most aggressive strains caused symptoms up to the third node. Real‐time quantitative PCR showed that fungal colonization reached the third node for all the tested species, but a low percentage of plants showed colonization above the third node following inoculation with the most aggressive strains. Fungal growth was detected in symptomless tissues but none of the three species was able to colonize as far as the head tissues. However, even if the pathogens were not detected in the heads, DON was detected in head tissues of the plants inoculated with the most aggressive strains. These results demonstrate that F. pseudograminearum, F. culmorum and F. graminearum, under the same experimental conditions, follow a similar pattern of symptom progression, fungal colonization and DON translocation after stem base infection. Differences in the extent of symptoms, fungal colonization and mycotoxin distribution were mainly attributable to strain aggressiveness. These findings provide comparative information on the events following infection of the stem base of wheat by three of the most important FCR casual agents.     

Elucidation of factors contributing to onset of Cercospora leaf blight during later reproductive development of soybean

In Louisiana, Cercospora leaf blight (CLB) of soybean is primarily caused by Cercospora cf. flagellaris, which can be detected in asymptomatic leaves as early as V3 (third trifoliate) growth stage, while symptoms appear around R5 (beginning seed). The fungus produces cercosporin, an important virulence factor, the in vitro production of which is significantly reduced in the absence of simple sugars. Our objective was to investigate the role of foliar simple sugars and endophytic bacteria in CLB symptom onset. C. cf. flagellaris was cultured in filter-sterilized, untreated extracts made from asymptomatic R2 and R5 leaves; in extracts treated with chloroform to isolate hydrophilic molecules, including simple sugars; or with antibiotics to assess the role of endophytic bacteria. Endophytic bacteria were also isolated from leaves and leaf extracts and their interaction with C. cf. flagellaris was investigated. The fungus produced more cercosporin in total R5 and simple sugar-amended R2 leaf extracts, than in R2 leaf extracts. Furthermore, it produced more cercosporin in chloroform-treated R5 leaf extracts, which contained significantly higher levels of simple sugars, especially fructose, than the R2. To our knowledge, this is the first reported connection between foliar sugar content and cercosporin production. Additionally, morphologically diverse bacteria were isolated from different stages of soybean development. Also, bacterial endophytes in R5 leaf extracts and Paenibacillus polymyxa, isolated from R2 leaves, significantly reduced the growth of C. cf. flagellaris. These findings implicated soybean leaf fructose in CLB development and highlighted the potential of using foliar bacterial endophytes for CLB management.     

Characterization of fluorescent Pseudomonas spp. associated with roots and soil of two sorghum genotypes

Sorghum is used as bioenergy feedstock, animal feed, and food. Economical methods for disease prevention and control are valuable for producers. Fluorescent Pseudomonas spp. were isolated from sorghum roots and surrounding soil with the goal of finding isolates that significantly inhibited sorghum fungal pathogens. Fluorescent pseudomonads were collected from seedlings of sorghum cultivars RTx433 and Redlan and wheat cultivar Lewjain, grown in two soils. Lewjain is known to support growth of producers of the antibiotic, 2,4-diacetylphloroglucinol (2,4-DAPG). Isolates from all three plants were assessed for hydrogen cyanide (HCN) and extracellular protease production, and for a 2,4-DAPG gene, phlD. Both soil type and plant type affected HCN- and protease-production, but phlD was not affected. Subsets of phlD ⁺ isolates were chosen to determine phlD genotypes and to conduct in vitro inhibition assays against sorghum pathogens. Most isolates from sorghum and wheat were genotype D, previously associated with superior root colonization. phlD ⁺ sorghum isolates were co-cultured with five sorghum pathogens. One isolate from each sorghum line exhibited inhibition to all five pathogens but more Redlan isolates were inhibitory to the virulent pathogen, Fusarium thapsinum, than RTx433 isolates. Nearly all inhibitory isolates from either sorghum cultivar were from one soil type. This is consistent with what had been previously observed in field studies: that soil type played a significant role in determining characteristics of fluorescent Pseudomonas spp. isolated from roots or soil, but sorghum genotype also had a considerable effect.     

Biocontrol of potato early blight and suppression of Alternaria grandis sporulation by Clonostachys spp.

Early blight, caused by Alternaria solani and A. grandis, severely affects potato crops around the world, and few management options apart from intensive fungicide use are available. In this work, we tested whether biocontrol treatments with different species of the mycoparasitic fungus Clonostachys could reduce early blight severity on the foliage of pot-grown potatoes of cultivar Ágata, and affect the survival and sporulation of the causal pathogen A. grandis. Twenty isolates from five Clonostachys species were screened in a preliminary biocontrol trial, and nine of them were selected and tested further in two confirmation trials. Treatments with three isolates from three species (C. chloroleuca, C. pseudochroleuca, C. rhizophaga) resulted in a significant reduction in disease severity in both confirmation trials, with control efficacy ranging from 88.7% to 92.9% in the first trial, and from 83.1% to 84.7% in the second trial. All Clonostachys isolates used in the confirmation trials survived on potato leaf tissues for at least 15 days, and resumed growth when subjected to high humidity, resulting in mycoparasitic overgrowth and significant reduction of the pathogen sporulation. Our results provide evidence that different species of Clonostachys, not only the ubiquitous C. rosea, are sources of useful candidates for the biocontrol of potato early blight and possibly other plant diseases caused by fungal pathogens.     

Identification and pathogenicity of Fusarium species associated with pokkah boeng of sugarcane in Brazil

Brazil is the world’s biggest producer of sugarcane (Saccharum spp. hybrids). Pokkah boeng is an important fungal disease in this crop caused by members of the Fusarium fujikuroi species complex (FFSC) and characterized by deformation of the aerial part of the plant and stem rot. While the occurrence of symptoms has been reported in plantations in Brazil, no official reports of the disease exist. In this study, species of the FFSC were identified that are associated with sugarcane plants with symptoms of pokkah boeng in Brazil. This was achieved using two-loci molecular phylogeny, sexual compatibility and analysis of morphological markers. The ability of strains to cause disease in plants of sugarcane, maize, sorghum and millet was also evaluated. The 39 isolates studied were identified as F. sacchari, F. proliferatum and another, still unknown, phylogenetic lineage that is sister to F. andiyazi. Crossing field isolates of F. sacchari and F. proliferatum with their respective tester strains produced fertile perithecia and viable ascospores. All three species induced symptoms of pokkah boeng on inoculated sugarcane plants and caused stem rot in maize, sorghum and millet. Symptoms on sugarcane are chlorosis and necrosis of leaves, punctured lesions, twisted leaves, reduction of the total leaf area, death of the top of the plant and stalk rot. The findings confirmed the aetiology of the disease in Brazil, generated basic knowledge for the development of strategies for diagnosis and monitoring of the disease and support breeding programmes for selecting resistant germplasm.     

Contrasting species boundaries between sections Alternaria and Porri of the genus Alternaria

The fungal genus Alternaria comprises a large number of asexual taxa with diverse ecological, morphological and biological modes ranging from saprophytes to plant pathogens. Understanding the speciation processes affecting asexual fungi is important for estimating biological diversity, which in turn affects plant disease management and quarantine enforcement. This study included 106 isolates of Alternaria representing five phylogenetically defined clades in two sister sub‐generic groups: section Porri (A. dauci, A. solani and A. limicola) and section Alternaria (A. alternata/tenuissima and A. arborescens). Species in section Porri are host‐specific while species in section Alternaria have wider host ranges. For each isolate, DNA sequences of three genes (Alt a1, ATPase, Calmodulin) were used to estimate phylogenies at the population and species levels. Three multilocus haplotypes were distinguished among A. dauci isolates and only one haplotype among A. solani and A. limicola isolates, revealing low or no differentiation within each taxon and strong clonal structure for taxa in this section. In contrast, 37 multilocus haplotypes were found among A. alternata/tenuissima isolates and 21 multilocus haplotypes among A. arborescens isolates, revealing much higher genotypic diversity and multiple clonal lineages within taxa, which is not typical of asexual reproducing lineages. A species tree was inferred using a Yule Speciation model and a strict molecular clock assumption. Species boundaries were well defined within section Porri. However, species boundaries within section Alternaria were only partially resolved with no well‐defined species boundaries, possibly due to incomplete lineage sorting. Significant association with host specificity seems a driving force for speciation.     

Endophytic Beauveria bassiana activates expression of defence genes in grapevine and prevents infections by grapevine downy mildew Plasmopara viticola

Fungal entomopathogens like Beauveria bassiana (Ascomycota: Hypocreales) are known as antagonists of insects with multiple functional and ecological roles, and have attracted increased attention as biocontrol agents in integrated pest management programmes. For some crop plants, it has been proven that endophytic B. bassiana, besides its entomopathogenic habit, can provide protection against plant pathogens or limit their damaging effects. However, for grapevine, limited knowledge is available on the influence of endophytic B. bassiana on fungal pathogens and about the mechanisms underlying putative protection effects. This study assessed the protective potential of endophytic B. bassiana against grapevine downy mildew Plasmopara viticola in greenhouse experiments. Three and seven days after a B. bassiana treatment, potted grapevine plants were inoculated with P. viticola and the evolving disease severity was assessed. Disease severity was significantly reduced in B. bassiana-treated plants compared to control plants, depending on the age of leaves. Furthermore, a microarray and an RT-qPCR analysis were performed to work out fundamental aspects of genes involved in the interaction between grapevine and B. bassiana. The results indicate an up-regulation of diverse defence-related genes in grapevine as a response to endophytic establishment of B. bassiana. Thus, endophytic establishment of an entomopathogenic fungus such as B. bassiana in grapevine plants would represent an alternative and sustainable plant protection strategy, with the potential for reducing pesticide applications in viticulture.