Botrytis species: relentless necrotrophic thugs or endophytes gone rogue?

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

The Quantitative Basis of the Arabidopsis Innate Immune System to Endemic Pathogens Depends on Pathogen Genetics

The most established model of the eukaryotic innate immune system is derived from examples of large effect monogenic quantitative resistance to pathogens. However, many host-pathogen interactions involve many genes of small to medium effect and exhibit quantitative resistance. We used the Arabidopsis-Botrytis pathosystem to explore the quantitative genetic architecture underlying host innate immune system in a population of Arabidopsis thaliana. By infecting a diverse panel of Arabidopsis accessions with four phenotypically and genotypically distinct isolates of the fungal necrotroph B. cinerea, we identified a total of 2,982 genes associated with quantitative resistance using lesion area and 3,354 genes associated with camalexin production as measures of the interaction. Most genes were associated with resistance to a specific Botrytis isolate, which demonstrates the influence of pathogen genetic variation in analyzing host quantitative resistance. While known resistance genes, such as receptor-like kinases (RLKs) and nucleotide-binding site leucine-rich repeat proteins (NLRs), were found to be enriched among associated genes, they only account for a small fraction of the total genes associated with quantitative resistance. Using publically available co-expression data, we condensed the quantitative resistance associated genes into co-expressed gene networks. GO analysis of these networks implicated several biological processes commonly connected to disease resistance, including defense hormone signaling and ROS production, as well as novel processes, such as leaf development. Validation of single gene T-DNA knockouts in a Col-0 background demonstrate a high success rate (60%) when accounting for differences in environmental and Botrytis genetic variation. This study shows that the genetic architecture underlying host innate immune system is extremely complex and is likely able to sense and respond to differential virulence among pathogen genotypes.     

A putative new genus of avian schistosome transmitted by Biomphalaria straminea (Gastropoda: Planorbidae) in Brazil,with a discussion on the potential involvement in human cercarial dermatitis

Human cercarial dermatitis (HCD) caused by avian schistosomes is an emerging health issue in different parts of the world. Nevertheless, parasite diversity, life cycle, and involvement in HCD remain poorly known or neglected in South America. Herein, we reported data obtained during a long-term malacological survey carried out in Pampulha Reservoir, an urban eutrophic waterbody from Brazil between 2009 and 2012. An ocellate brevifurcate cercaria emerged from 55 of 16,235 (0.34%) specimens of Biomphalaria straminea. Samples of the cercariae were subjected to morphological, experimental, and molecular study (analysis of partial sequences of nuclear 28S and mitochondrial cox1 genes). The molecular analysis revealed that the larva corresponds to an avian schistosome; however, it does not correspond to any named genus. A close related isolate was previously reported in Biomphalaria sudanica from Kenya (molecular divergences of 0.54% and 9.62% for 28S and cox1, respectively). The morphology of this cercaria was compared with other avian schistosome larvae from Biomphalaria spp. Attempts to infect experimentally ducks (Cairina moschata) and mice revealed cutaneous manifestations after exposure to cercariae, but adult parasites were not obtained in these hosts. Phylogenetic analysis suggests this parasite is a putative new genus and species of avian schistosome. The potential involvement of the larvae herein described in cases of HCD in Brazil cannot be ruled out. Surprisingly, HCD was not reported in the country so far, which can be related to difficulties in its diagnosis in areas of overlap with human schistosomes.     

Molecular phylogeny and host use evolution of the genus Exorista Meigen (Diptera: Tachinidae)

Members of the genus Exorista are parasitoids of a diverse array of insect hosts in the orders, Lepidoptera, Hymenoptera, Mantodea and Orthoptera. Phylogenetic relationships among subgenera and species of Exorista were inferred using four nuclear (Tpi, white, 18S and 28S) and four mitochondrial DNA (16S, 12S, ND5 and CO1) genes in maximum parsimony (MP), maximum likelihood (ML) and Bayesian Markov chain Monte Carlo (MCMC) analyses. Separate trees based on different sets of genes (mt DNA, nuclear, ribosomal, etc.) were compared and found to be nearly concordant. According to the molecular tree generated from the concatenated sequence data, the genus Exorista is paraphyletic. The phylogenetic analyses indicate the existence of two major clades of Exorista, including two genera Parasetigena and Phorocera. Morphological traits supporting clades indicated by molecular analyses within this genus are evaluated. Evolutionary patterns of the host use and host shifts are examined by optimizing host information using maximum likelihood on the molecular phylogeny. The ancestral host group of the tribe Exoristini (excluding Ctenophorinia and Phorinia) appears to be the order Lepidoptera, although hosts of some species are unknown. A major host shift to the Hymenoptera occurred in the clade of subgenus Adenia, and the ancestral state of subgenus Spixomyia is equivocal because there is little information available on the hosts in members of a subclade of this group (subclade A: Exorista hyalipennis group).     

Botrytis sinoallii: a new species of the grey mould pathogen on Allium crops in China

A study was conducted to identify Botrytis spp. isolated from bulb onion, green onions, garlic, and garlic chives grown in Hubei Province of China. Based on colony morphology and conidial characteristics, 367 strains of Botrytis spp. were identified as five distinct species, namely, B. cinerea, B. squamosa, B. porri, B. byssoidea, and an undescribed Botrytis species (Botrytis sp.), which accounted for 64.3, 29.9, 3.3, 0.3, and 2.2%, respectively. The previously undescribed species is herein described as a new species, B. sinoallii sp. nov., which is characterized by production of numerous small sclerotia on potato dextrose agar. Phylogenetic analysis using partial sequences of three nuclear genes (G3PDH, HSP60, and RPB2) showed that B. sinoallii forms a unique lineage, which is closely related to B. squamosa, a well-known species on Allium crops, but distantly related to the other species of Botrytis on Allium crops, including B. cinerea, B. porri, B. aclada, B. allii, B. byssoidea, B. globosa, and B. sphaerosperma. Results of inoculation tests showed that B. sinoallii is a newly identified agent that can cause leaf blight of green onion, garlic, and garlic chives. Potential impact of B. sinoallii on production of Allium crops in China is discussed.     

Comparison of Botrytis cinerea populations isolated from two open-field cultivated host plants

The necrotrophic fungus Botrytis cinerea is reported to infect more than 220 host plants worldwide. In phylogenetical–taxonomical terms, the pathogen is considered a complex of two cryptic species, group I and group II. We sampled populations of B. cinerea on sympatric strawberry and raspberry cultivars in the North-East of Hungary for three years during flowering and the harvest period. Four hundred and ninety group II B. cinerea isolates were analyzed for the current study. Three different data sets were generated: (i) PCR-RFLP patterns of the ADP-ATP translocase and nitrate reductase genes, (ii) MSB1 minisatellite sequence data, and (iii) the fragment sizes of five microsatellite loci. The structures of the different populations were similar as indicated by Nei's gene diversity and haplotype diversity. The F statistics (F_st, G_st), and the gene flow indicated ongoing differentiation within sympatric populations. The population genetic parameters were influenced by polymorphisms within the three data sets as assessed using Bayesian algorithms. Data Mining analysis pointed towards the five microsatellite loci as the most defining markers to study differentiation in the 490 isolates. The results suggest the occurrence of host-specific, sympatric divergence of generalist phytoparasites in perennial hosts.     

Molecular identification of Botrytis cinerea,Botrytis paeoniae and Botrytis pseudocinerea associated with gray mould disease in peonies (Paeonia lactiflora Pall.) in Southern Chile

BackgroundIn Chile, the peony is the most important ornamental flower exported from the country. Gray mould is a phytopathological problem of this crop. This disease is caused by Botrytis cinerea and Botrytis paeoniae.AimsWe carried out the first survey of Botrytis species associated with peony gray mould in Southern Chile to estimate the diversity of these pathogens.MethodsDiseased peony leaves were collected from seven locations in Southern Chile covering a distance of 300 km. The Botrytis isolates obtained were studied by morphological and molecular methods. Finally, a PCR assay using primers based on the necrosis and ethylene-inducing protein gene (nep1) was used to specifically identify B. paeoniae.ResultsSeventeen isolates belonging to Botrytis genus were obtained, and all of them were pathogenic to peonies when inoculated in plants grown in a greenhouse. Morphological analyses showed that four isolates shared common characteristics, which distinguish them from the rest. Homology and phylogenetic analysis of G3PDH, as well as determination of the Bc-hch allele, allowed us to identify 12 isolates as B. cinerea, 4 as B. paeoniae and one isolate as Botrytis pseudocinerea. The PCR assay was found to be specific to B. paeoniae, amplifying a single band of 470 bp.ConclusionsThree Botrytis species involved in peony gray mould disease are present in Chile. This is the first time that both B. paeoniae and B. pseudocinerea have been reported to be present in the country and also that they affect peonies. Finally, to our knowledge, the PCR based method herein described is the first of its kind to be used to identify B. paeoniae.     

Cytochrome c oxydase I phylogenetic analysis of Haemogregarina parasites (Apicomplexa,Coccidia, Eucoccidiorida,Haemogregarinidae) confirms the presence of three distinct species within the freshwater turtles of Tunisia

Species of Haemogregarina are apicomplexan blood parasites that use vertebrates as intermediate hosts. Due to limited interspecific morphological characters within the genus during the last decade, 18S rRNA gene sequences were widely used for species identification. As coinfection patterns were recently reported from nuclear molecular data for two sympatric freshwater turtles Mauremys leprosa and Emys orbicularis from Tunisia, our objectives were to design COI specific primers to confirm the presence of three distinct species in both host species. Blood samples were collected from 22 turtles, from which DNAs were extracted and used as templates for amplification. Following different rounds of PCR and nested PCR, we designed specific Haemogregarina COI primers that allowed the sequencing of nine distinct haplotypes. Phylogenetic Bayesian analysis revealed the occurrence of three well-differentiated sublineages that clustered together into a single clade. Based on pairwise genetic distances (p-distance), we confirmed the occurrence of three distinct but phylogenetically closely related species coinfecting M. leprosa and E. orbicularis in the same aquatic environments. Our results demonstrate that the use of fast evolving genes within Haemogregarina will help to investigate the parasite diversity within both intermediate vertebrate and definitive invertebrate hosts, and to assess the evolution, historical biogeography and specificity of haemogregarines.     

An Interspecies Comparative Analysis of the Predicted Secretomes of the Necrotrophic Plant Pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Phytopathogenic fungi form intimate associations with host plant species and cause disease. To be successful, fungal pathogens communicate with a susceptible host through the secretion of proteinaceous effectors, hydrolytic enzymes and metabolites. Sclerotinia sclerotiorum and Botrytis cinerea are economically important necrotrophic fungal pathogens that cause disease on numerous crop species. Here, a powerful bioinformatics pipeline was used to predict the refined S. sclerotiorum and B. cinerea secretomes, identifying 432 and 499 proteins respectively. Analyses focusing on S. sclerotiorum revealed that 16% of the secretome encoding genes resided in small, sequence heterogeneous, gene clusters that were distributed over 13 of the 16 predicted chromosomes. Functional analyses highlighted the importance of plant cell hydrolysis, oxidation-reduction processes and the redox state to the S. sclerotiorum and B. cinerea secretomes and potentially host infection. Only 8% of the predicted proteins were distinct between the two secretomes. In contrast to S. sclerotiorum, the B. cinerea secretome lacked CFEM- or LysM-containing proteins. The 115 fungal and oomycete genome comparison identified 30 proteins specific to S. sclerotiorum and B. cinerea, plus 11 proteins specific to S. sclerotiorum and 32 proteins specific to B. cinerea. Expressed sequence tag (EST) and proteomic analyses showed that 246 S. sclerotiorum secretome encoding genes had EST support, including 101 which were only expressed in vitro and 49 which were only expressed in planta, whilst 42 predicted proteins were experimentally proven to be secreted. These detailed in silico analyses of two important necrotrophic pathogens will permit informed choices to be made when candidate effector proteins are selected for function analyses in planta.     

First Report of Grey Mould Disease on Abelmoschus esculentus Caused by Botrytis cinerea in China

Okra (Abelmoschus esculentus), or Lady's finger, is a plant originating from Africa and is grown as a multipurpose crop in Asia, America and Europe nowadays. Grey mould disease was observed on okras in several okra fields in Ledong County, Hainan Province, and Shunyi District, Beijing City, during September to December in 2014 with the incidence varying from 20 to 55%. To identify the pathogen, the detailed morphological, cultural characteristics, pathogenicity test along with the DNA sequences for four gene regions ITS, G3PDH, HSP60 and RPB2 of our isolate were compared with those of some previously reported Botrytis cinerea and other similar species of the genus Botrytis. Results suggested this is the first report of grey mould disease on Abelmoschus esculentus caused by Botrytis cinerea in China.     

Botrytis Disease of Tea in Turkey

Botrytis disease of tea was reported for the first time from Rize, Turkey. The causal agent was identified as Botrytis cinerea based on morphological and cultural characteristics. Also, the species‐specific PCR assays confirmed the identification of all B. cinerea isolates. The pathogen caused blight of shoots, buds, flowers and young leaves, shoot canker and leaf spots. The disease was observed in Rize central district and Derepazar?, Çaml?hem?in, Çayeli, Pazar, Hem?in, ?kizdere, Arde?en and ?yidere districts.     

Modes of acquisition of Wolbachia: horizontal transfer, hybrid introgression, and codivergence in the Nasonia species complex

Wolbachia are maternally inherited bacteria that infect a large number of insects and are responsible for different reproductive alterations of their hosts. One of the key features of Wolbachia biology is its ability to move within and between host species, which contributes to the impressive diversity and range of infected hosts. Using multiple Wolbachia genes, including five developed for Multi-Locus Sequence Typing (MLST), the diversity and modes of movement of Wolbachia within the wasp genus Nasonia were investigated. Eleven different Wolbachia were found in the four species of Nasonia , including five newly identified infections. Five infections were acquired by horizontal transmission from other insect taxa, three have been acquired by hybridization between two Nasonia species, which resulted in a mitochondrial- Wolbachia sweep from one species to the other, and at least three have codiverged during speciation of their hosts. The results show that a variety of transfer mechanisms of Wolbachia are possible even within a single host genus. Codivergence of Wolbachia and their hosts is uncommon and provides a rare opportunity to investigate long-term Wolbachia evolution within a host lineage. Using synonymous divergence among codiverging infections and host nuclear genes, we estimate Wolbachia mutation rates to be approximately one-third that of the nuclear genome.     

Cloning and Partial Characterization of Endopolygalacturonase Genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

Diversity and species boundaries in floricolous downy mildews

Floricolous downy mildews are a monophyletic group of members of the genus Peronospora (Oomycota, Peronosporales). These downy mildews can be found on a variety of families of the Asteridae, including Asteraceae, Campanulaceae, Dipsacaceae, Lamiaceae, and Orobanchaceae. With the exception of Peronospora radii, which can also cause economically relevant losses, sporulation usually takes place only on floral parts of their hosts. However, only very few specimens of these mostly inconspicuous downy mildews have so far been included in molecular phylogenies. Focusing on Lamiaceae, we have investigated multiple specimens of floricolous downy mildews for elucidating species boundaries and host specificity in this group. Based on both mitochondrial and nuclear loci, it became apparent that phylogenetic lineages in the Lamiaceae seem to be host genus specific and significant sequence diversity could be found between lineages. Based on distinctiveness in both phylogenetic reconstructions and morphology, the downy mildew on flowers of Stachys palustris is introduced as a new species, Peronospora jagei sp. nov., which can be morphologically distinguished from Peronospora stigmaticola by broader and shorter conidiospores. The diversity of the floricolous down mildews might be higher than previously assumed, although specimens from a much broader set of samples will be needed to confirm this view.     

The large mimosoid genus Inga Mill. (tribe Ingeae,Caesalpinioideae) is nodulated by diverse Bradyrhizobium strains in its main centers of diversity in Brazil

Inga (Caesalpinioideae) is the type genus of the Ingeae tribe in the mimosoid clade. It comprises about 300 species, all trees or treelets, and has an exclusively neotropical distribution, with Brazil as its main center of diversity. In this study, we analyzed the diversity of 40 strains of rhizobia isolated from root nodules collected from ten species of Inga belonging to different types of vegetation in Brazil. Sequences of their housekeeping genes (dnaK, recA, rpoB, gyrB and glnII), 16S rRNA genes, internal transcribed spacer (ITS) regions, as well as their symbiosis-essential genes (nodC and nifH) were used to characterize them genetically. The ability of the rhizobia to form nodules on Inga spp., and on the promiscuous legume siratro (Macroptilium atropurpureum) was also evaluated. A multilocus sequence analysis (MLSA) combined with an analysis of the ITS region showed that the isolates were distributed into four main groups (A-D) within the large genus Bradyrhizobium. Analysis of the nodC and nifH genes showed that the isolates formed a separate branch from all described species of Bradyrhizobium, except for B. ingae. Most of the tested isolates formed nodules on siratro and all isolates tested nodulated Inga spp. Our results suggest a unique co-evolutionary history of Bradyrhizobium and Inga and demonstrate the existence of potential new species of microsymbionts nodulating this important and representative genus of leguminous tree from the Caesalpinioideae mimosoid clade.     

Phylogeography of Rhabdias spp. (Nematoda: Rhabdiasidae) collected from Bufo species in Honshu,Shikoku, and Kyushu,Japan including possible cryptic species

The genus Rhabdias Stiles & Hassall, 1905 comprises lung parasites of amphibians and reptiles worldwide. In Japan, 9 species have been recorded, including Rhabdias incerta Wilkie, 1930 which has been reported only in Bufo species. In this study, to assess the diversity of R. incerta, we performed molecular analyses of Rhabdias species sampled from three species/subspecies of Japanese toads namely Bufo japonicus, B. japonicus formosus, and B. torrenticola, collected in various regions of Honshu, Shikoku, and Kyushu, Japan. DNA sequence divergence was compared using mtDNA (COI) and nuclear DNA (28S) to identify possible cryptic species. Morphological analysis was performed through light microscopy and scanning electron microscopy (SEM). The results revealed that Bufo spp. serve as hosts for at least three Rhabdias species. Morphologically, most samples were identified as R. incerta but with a longer body and larger buccal cavity than originally described. Rhabdias incerta appears to be specific to the genus Bufo and is further subdivided into two or three phylogroups based on subspecies divisions and biogeography of their host. Some Rhabdias specimens collected in this study resemble R. tokyoensis Wilkie, 1930, parasitic in hosts from the order Caudata, which suggests host switching. Both molecular and morphological analyses suggested the presence of undescribed and cryptic Rhabdias species within toads collected in Japan. This study was the first to molecularly characterize Rhabdias species in Japan, including novel sequences of R. incerta and two undescribed species.     

Comparative Genomic Analysis of 45 Type Strains of the Genus Bifidobacterium: A Snapshot of Its Genetic Diversity and Evolution

Bifidobacteria are well known for their human health-promoting effects and are therefore widely applied in the food industry. Members of the Bifidobacterium genus were first identified from the human gastrointestinal tract and were then found to be widely distributed across various ecological niches. Although the genetic diversity of Bifidobacterium has been determined based on several marker genes or a few genomes, the global diversity and evolution scenario for the entire genus remain unresolved. The present study comparatively analyzed the genomes of 45 type strains. We built a robust genealogy for Bifidobacterium based on 402 core genes and defined its root according to the phylogeny of the tree of bacteria. Our results support that all human isolates are of younger lineages, and although species isolated from bees dominate the more ancient lineages, the bee was not necessarily the original host for bifidobacteria. Moreover, the species isolated from different hosts are enriched with specific gene sets, suggesting host-specific adaptation. Notably, bee-specific genes are strongly associated with respiratory metabolism and are potential in helping those bacteria adapt to the oxygen-rich gut environment in bees. This study provides a snapshot of the genetic diversity and evolution of Bifidobacterium, paving the way for future studies on the taxonomy and functional genomics of the genus.     

New host,geographic records,and histopathologic studies of Angiostrongylus spp (Nematoda: Angiostrongylidae) in rodents from Argentina with updated summary of records from rodent hosts and host specificity assessment

To date, 21 species of the genus Angiostrongylus (Nematoda: Angiostrongylidae) have been reported around the world, 15 of which are parasites of rodents. In this study, new host, geographic records, and histopathologic studies of Angiostrongylus spp in sigmodontine rodents from Argentina, with an updated summary of records from rodent hosts and host specificity assessment, are provided. Records of Angiostrongylus costaricensis from Akodon montensis andAngiostrongylus morerai from six new hosts and geographical localities in Argentina are reported. The gross and histopathologic changes in the lungs of the host species due to angiostrongylosis are described. Published records of the genus Angiostrongylus from rodents and patterns of host specificity are presented. Individual Angiostrongylusspecies parasitise between one-19 different host species. The most frequent values of the specificity index (STD) were between 1-5.97. The elevated number of host species (n = 7) of A. morerai with a STD = 1.86 is a reflection of multiple systematic studies of parasites from sigmodontine rodents in the area of Cuenca del Plata, Argentina, showing that an increase in sampling effort can result in new findings. The combination of low host specificity and a wide geographic distribution of Angiostrongylus spp indicates a troubling epidemiological scenario although, as yet, no human cases have been reported.