Plant Pathogenic Species Research Articles

Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III-secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus.

SummaryThe cucurbit pathogenic bacterium Acidovorax citrulli requires a functional type III secretion system (T3SS) for pathogenicity. In this bacterium, as with Xanthomonas and Ralstonia spp., an AraC‐type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III‐secreted effectors (T3Es). The annotation of a sequenced A. citrulli strain revealed 11 T3E genes. Assuming that this could be an underestimation, we aimed to uncover the T3E arsenal of the A. citrulli model strain, M6. Thorough sequence analysis revealed 51 M6 genes whose products are similar to known T3Es. Furthermore, we combined machine learning and transcriptomics to identify novel T3Es. The machine‐learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA‐Seq revealed differential gene expression between wild‐type M6 and a mutant defective in HrpX: 159 and 28 genes showed significantly reduced and increased expression in the mutant relative to wild‐type M6, respectively. Data combined from these approaches led to the identification of seven novel T3E candidates that were further validated using a T3SS‐dependent translocation assay. These T3E genes encode hypothetical proteins that seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study places A. citrulli among the ‘richest’ bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.

The Mode of Action of Bacillus Species against Fusarium graminearum, Tools for Investigation, and Future Prospects.

Fusarium graminearum is a pervasive plant pathogenic fungal species. Biological control agents employ various strategies to weaken their targets, as shown by Bacillus species, which adopt various mechanisms, including the production of bioactive compounds, to inhibit the growth of F. graminearum. Various efforts to uncover the antagonistic mechanisms of Bacillus against F. graminearum have been undertaken and have yielded a plethora of data available in the current literature. This perspective article attempts to provide a unified record of these interesting findings. The authors provide background knowledge on the use of Bacillus as a biocontrol agent as well as details on techniques and tools for studying the antagonistic mechanism of Bacillus against F. graminearum. Emphasizing its potential as a future biological control agent with extensive use, the authors encourage future studies on Bacillus as a useful antagonist of F. graminearum and other plant pathogens. It is also recommended to take advantage of the newly invented analytical platforms for studying biochemical processes to understand the mechanism of action of Bacillus against plant pathogens in general.

Zearalenone and ß-Zearalenol But Not Their Glucosides Inhibit Heat Shock Protein 90 ATPase Activity.

The mycotoxin zearalenone (ZEN) is produced by many plant pathogenic Fusarium species. It is well known for its estrogenic activity in humans and animals, but whether ZEN has a role in plant–pathogen interaction and which process it is targeting in planta was so far unclear. We found that treatment of Arabidopsis thaliana seedlings with ZEN induced transcription of the AtHSP90.1 gene. This heat shock protein (HSP) plays an important role in plant–pathogen interaction, assisting in stability and functionality of various disease resistance gene products. Inhibition of HSP90 ATPase activity impairs functionality. Because HSP90 inhibitors are known to induce HSP90 gene expression and due to the structural similarity with the known HSP90 inhibitor radicicol (RAD), we tested whether ZEN and its phase I metabolites α- and ß-zearalenol are also HSP90 ATPase inhibitors. Indeed, AtHSP90.1 and wheat TaHSP90-2 were inhibited by ZEN and ß-zearalenol, while α-zearalenol was almost inactive. Plants can efficiently glycosylate ZEN and α/ß-zearalenol. We therefore tested whether glucosylation has an effect on the inhibitory activity of these metabolites. Expression of the A. thaliana glucosyltransferase UGT73C6 conferred RAD resistance to a sensitive yeast strain. Glucosylation of RAD, ZEN, and α/ß-zearalenol abolished the in vitro inhibitory activity with recombinant HSP90 purified from Escherichia coli. In conclusion, the mycotoxin ZEN has a very prominent target in plants, HSP90, but it can be inactivated by glycosylation. This may explain why there is little evidence for a virulence function of ZEN in host plants.

Climate change and plant pathogen biodiversity: The case of witches’ broom outbreak on Vachellia xanthophloea (Benth.) Banfi & Galasso) in Botswana

Climate change, typified by drought, floods and rising temperatures continue to affect biodiversity in the world. Recently, outbreaks of new insect, weed and plant pathogen species have been reported in SADC and some old ones have increased in severity while others have diminished. Examples of new insects that have threated food security and trade in the region include the fall army worm (Spodoptera frugiperda) and tomato leaf miner (Tuta absoluta). Over 50% of the Hartbeespoort Dam near Pretoria is covered by water hyacinth (Eichhornia crassipes) reducing property values and tourism in the area. In August 2014, witches’ broom outbreak on fever tree was first observed in Gaborone. Microscopic examination of disease signs revealed, urediospores, teliospores, aeciospores and aecia typical of Ravenelia sp. Samples were sent to the FABI, University of Pretoria where the pathogen was tentatively identified as R. natalensis or R. hieronymi. After the 2014/2015 and 2015/16 poor rain seasons few new infections were recorded in August. Following the heavy rains caused by cyclone Dineo, the epiphytotic exploded starting July 2018 and almost all fever trees in the major towns of Botswana were attacked. The witches’ brooms were also infected by powdery mildew (Erysiphe sp.) and Diplodia sp. which caused black twig dieback. The fever tree natural distribution is limited to the Tuli block but it has been introduced south as a landscape and shade tree. The tree is now invasive spreading rapidly along drainage channels and rivers around Gaborone. Currently the pathogen may be a threat to other Vachellia and Senegalia spp. in Botswana. The witches’ brooms are releasing trillions of spores into the air which may result in new human allergies. The implications of the outbreak on tree biodiversity and how the disease can be managed should be investigated.

Osmotolerance as a determinant of microbial ecology: A study of phylogenetically diverse fungi

Osmotic stress induced by high solute concentration can prevent fungal metabolism and growth due to alterations in properties of the cytosol, changes in turgor, and the energy required to synthesize and retain compatible solutes. We used germination to quantify tolerance/sensitivity to the osmolyte KCl (0.1–4.5 M, in 0.1 M increments) for 71 strains (40 species) of ecologically diverse fungi. These include 11 saprotrophic species (17 strains, including two xerophilic species), five mycoparasitic species (five strains), six plant-pathogenic species (13 strains), and 19 entomopathogenic species (36 strains). A dendrogram obtained from cluster analyses, based on KCl inhibitory concentrations 50 % and 90 % calculated by Probit Analysis, revealed three groups of fungal isolates accordingly to their osmotolerance. The most-osmotolerant group (Group 3) contained the majority of saprotrophic fungi, and Aspergillus niger (F19) was the most tolerant. The highly xerophilic Aspergillus montevidense and Aspergillus pseudoglaucus were the second- and third-most tolerant species, respectively. All Aspergillus and Cladosporium species belonged to Group 3, followed by the entomopathogens Colletotrichum fioriniae, Simplicillium lanosoniveum, and Trichothecium roseum. Group 2 exhibited a moderate osmotolerance, and included plant-pathogens such as Colletotrichum and Fusarium, mycoparasites such as Clonostachys spp, some saprotrophs such as Mucor and Penicillium spp., and some entomopathogens such as Isaria, Lecanicillium, Mariannaea, Simplicillium, and Torrubiella. Group 1 contained the osmo-sensitive strains: the rest of the entomopathogens and the mycoparasitic Gliocladium and Trichoderma. Although stress tolerance did not correlate with their primary ecological niche, classification of these 71 fungal strains was more closely aligned with their ecology than with their phylogenetic relatedness. We discuss the implications for both microbial ecology and fungal taxonomy.

Different Combretum molle (Combretaceae) leaf extracts contain several different antifungal and antibacterial compounds

Combretum molle occurs widely in Africa and is used traditionally to treat many different ailments including microbial infections. Some work has been done on the antimicrobial activity of bark extracts, but not on leaf extracts, a sustainable resource.Dried ground leaves were extracted with methanol, butanol, acetone, ethyl acetate, chloroform, dichloromethane and n-hexane. The antibacterial and antifungal activities were determined by using a serial microplate dilution method and the number of antimicrobial compounds were determined by bioautography. Both animal (Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus) and plant (Penicillium janthinellum, Aspergillus niger, Aspergillus parasiticus, Fusarium oxysporum, Rhizoctonia solani) pathogenic fungal species were examined. Four important nosocomial bacterial species (Staphylococcus aureus, Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa) were also examined.Butanol extracted the highest mass of plant material (41 mg) and hexane and dichloromethane extracted the lowest masses, 8 and 9 mgl, respectively. In bioautograms developed with BEA sprayed with Aspergillus fumigatus, all the extracts had clear zones of inhibition at R values of c. 0.12, 0.23, and 0.40 for the different fungi.The MIC values of the extracts ranged from 0.04 to 0.77 mg/ml after 12 h incubation. The acetone leaf extract was the most active with an MIC of 0.04 mg/ml against P. janthinellum, an important plant fungal pathogen. The total activity of 775 ml/g (indicating that the extract obtained from 1 g of leaves can be diluted to 775 ml and will still inhibit the growth) means that the acetone leaf extract could be used to control P. janthinellum infections on plants.

Epitypification of Cercospora rautensis, the causal agent of leaf spot disease on Securigera varia, and its first report from Iran.

Cercospora is a well-studied and important genus of plant pathogenic species responsible for leaf spots on a broad range of plant hosts. The lack of useful morphological traits and the high degree of variation therein complicate species identifications in Cercospora. Recent studies have revealed multi-gene DNA sequence data to be highly informative for species identification in Cercospora. During the present study, Cercospora isolates obtained from Crownvetch (Securigera varia) in Iran and Romania were subjected to an eight-gene (ITS, tef1, actA, cmdA, his3, tub2, rpb2 and gapdh) analysis. By applying a polyphasic approach including morphological characteristics, host data, and molecular analyses, these isolates were identified as C. rautensis. To our knowledge, this is the first record of C. rautensis from Iran (Asia). In addition, an epitype is designated here for C. rautensis.

Comparative genomics of plant pathogenic Botrytis species with distinct host specificity

BackgroundFungi of the genus Botrytis (presently containing ~ 35 species) are able to infect more than 1400 different plant species and cause losses in a wide range of crops of economic importance. The best studied species is B. cinerea, which has a broad host range and is one of the best studied necrotrophic plant pathogenic fungi. Most other Botrytis spp. have a narrow host range and have been studied in less detail. To characterize genomic variation among different representatives of Botrytis spp., we sequenced and annotated the draft genomes of nine Botrytis species: B. calthae, B. convoluta, B. elliptica, B. galanthina, B. hyacinthi, B. narcissicola, B. paeoniae, B. porri and B. tulipae.ResultsBioinformatics and comparative genomics tools were applied to determine a core of 7668 shared protein families in all Botrytis species, which grouped them in two distinct phylogenetic clades. The secretome of all nine Botrytis spp. was similar in number (ranging from 716 to 784 predicted proteins). A detailed analysis of the molecular functions of the secretome revealed that shared activities were highly similar. Orthologs to effectors functionally studied in B. cinerea were also present in the other Botrytis species. A complex pattern of presence/absence of secondary metabolite biosynthetic key enzymes was observed.ConclusionsComparative genomics of Botrytis show that overall, species share the main signatures and protein families in the secreted proteins, and of known effectors. Our study provides leads to study host range determinants in the genus Botrytis and provides a stepping stone to elucidate the roles of effector candidates in the infection process of these species.

Experimental infection in mice with Erwinia persicina

Erwinia persicinus (E. persicina) is a plant pathogenic bacterial species that was previously isolated from a case of human infection. This study aimed to create an experimental infection protocol for E. persicina in laboratory mice. Seventy-two adult mice were divided into four groups (18 animal/group): the control group (G1), the group infected with E. persicina (G2), the group immune-suppressed with cyclophosphamide (G3) and the group immune-suppressed with cyclophosphamide and infected with E. persicina (G4). G2 and G4 were injected with 200 μL of (1 × 1013 cfu/ml) concentration intraperitoneally. Clinical signs, such as diarrhoea, apathy and mortality were observed only in G2 and G4 animals. E. persicina was not detected in blood. Necropsies of the G2 and G4 animals showed lesions in the intestine, liver, kidney and lung tissue. These lesions were characterized by infiltration of inflammatory cells, hyperaemia and focal areas of tissue necrosis in the liver. The results of the pro-inflammatory cytokines analysis revealed a significant increase in the levels of TNF-α and IL1-β in the liver tissue of the G4 group. E. persicina is an emerging bacterium that can cause pathological lesions into mammalian tissue, which warrants further investigation.

Phenamacril is a reversible and noncompetitive inhibitor of Fusarium class I myosin

The cyanoacrylate compound phenamacril (also known as JS399-19) is a recently identified fungicide that exerts its antifungal effect on susceptible Fusarium species by inhibiting the ATPase activity of their myosin class I motor domains. Although much is known about the antifungal spectrum of phenamacril, the exact mechanism behind the phenamacril-mediated inhibition remains to be resolved. Here, we describe the characterization of the effect of phenamacril on purified myosin motor constructs from the model plant pathogen and phenamacril-susceptible species Fusarium graminearum, phenamacril-resistant Fusarium species, and the mycetozoan model organism Dictyostelium discoideum Our results show that phenamacril potently (IC50 ∼360 nm), reversibly, and noncompetitively inhibits ATP turnover, actin binding during ATP turnover, and motor activity of F. graminearum myosin-1. Phenamacril also inhibits the ATPase activity of Fusarium avenaceum myosin-1 but has little or no inhibitory effect on the motor activity of Fusarium solani myosin-1, human myosin-1c, and D. discoideum myosin isoforms 1B, 1E, and 2. Our findings indicate that phenamacril is a species-specific, noncompetitive inhibitor of class I myosin in susceptible Fusarium sp.

Didymella eriobotryae sp. nov. (Didymellaceae) and Arthrinium arundinis (Apiosporaceae) from fruit of Eriobotrya japonica (loquat) in China

Arthrinium (Apiosporaceae) and Didymella (Didymellaceae) are species-rich genera and most of the members in these genera are plant pathogenic, saprobic or endophytic species that have been recorded from a wide range of hosts. The present study introduces Didymella eriobotryae sp. nov. and a new record of Arthrinium arundinis which are associated with decaying fruit of Eriobotrya japonica in China. Morphology and DNA sequence data are employed to determine the taxonomic placement of these species.

Comparative evolutionary histories of fungal proteases reveal gene gains in the mycoparasitic and nematode-parasitic fungus Clonostachys rosea

BackgroundThe ascomycete fungus Clonostachys rosea (order Hypocreales) can control several important plant diseases caused by plant pathogenic fungi and nematodes. Subtilisin-like serine proteases are considered to play an important role in pathogenesis in entomopathogenic, mycoparasitic, and nematophagous fungi used for biological control. In this study, we analysed the evolutionary histories of protease gene families, and investigated sequence divergence and regulation of serine protease genes in C. rosea.ResultsProteases of selected hypocrealean fungal species were classified into families based on the MEROPS peptidase database. The highest number of protease genes (590) was found in Fusarium solani, followed by C. rosea with 576 genes. Analysis of gene family evolution identified non-random changes in gene copy numbers in the five serine protease gene families S1A, S8A, S9X, S12 and S33. Four families, S1A, S8A, S9X, and S33, displayed gene gains in C. rosea. A gene-tree / species-tree reconciliation analysis of the S8A family revealed that the gene copy number increase in C. rosea was primarily associated with the S08.054 (proteinase K) subgroup. In addition, regulatory and predicted structural differences, including twelve sites evolving under positive selection, among eighteen C. rosea S8A serine protease paralog genes were also observed. The C. rosea S8A serine protease gene prs6 was induced during interaction with the plant pathogenic species F. graminearum.ConclusionsNon-random increases in S8A, S9X and S33 serine protease gene numbers in the mycoparasitic species C. rosea, Trichoderma atroviride and T. virens suggests an involvement in fungal-fungal interactions. Regulatory and predicted structural differences between C. rosea S8A paralogs indicate that functional diversification is driving the observed increase in gene copy numbers. The induction of prs6 expression in C. rosea during confrontation with F. graminearum suggests an involvement of the corresponding protease in fungal-fungal interactions. The results pinpoint the importance of serine proteases for ecological niche adaptation in C. rosea, including a potential role in the mycoparasitic attack on fungal prey.

The current status on secondary metabolites produced by plant pathogenic Colletotrichum species

The fungal genus Colletotrichum contains about 190 species, many of which are responsible for serious plant diseases including those of commercial crops. These species infect a wide range of crops in the tropical, sub-tropical and temperate regions of the world. The diseases caused by Colletotrichum species are known as “anthracnose diseases” and are characterized by sunken necrotic tissue in which masses of orange conidia are produced. A significant number of the metabolites of Colletotrichum species are known to contribute to their pathogenicity. These phytotoxic metabolites when applied to the leaves of their host plants, induced symptoms which were similar to those of the anthracnose caused by the fungus. These metabolites have been shown to play a significant role in the mechanism of infection and pathogenesis. This review deals with the structures and biological activity of the secondary metabolites which have been isolated from these economically important filamentous fungi. The literature up to July 2018 is reviewed and 160 references are cited.

Biochemical Properties and Catalytic Specificity of a Novel Neutral Serine Peptidase Secreted by Fungus Pyrenochaetopsis sp.

The fungal genus Pyrenochaetopsis has received particular attention because of its different lifestyles, such as numerous plant pathogenic, saprophytic, and endophytic species. Its ability to infect plant cells relies heavily upon secreted peptidases. Here, we investigated the biochemical properties and catalytic specificity of a new serine peptidase secreted by the filamentous fungus Pyrenochaetopsis sp. We found that while this neutral serine peptidase displayed optimal activity at a pH of 7.0 and temperature of 45°C, it tolerated a wide range of pH conditions and temperatures lower than 45°C. Its peptidase activity was depressed by some metallic ions (such as aluminum, cobalt, and copper (II) chloride) and enhanced by others (such as sodium, lithium, magnesium, potassium, calcium, and manganese). Lastly, the enzyme showed the greatest specificity for non-polar amino acids, particularly leucine and isoleucine, and moderate specificity for basic and neutral polar amino acids. It displayed the least specificity for acidic residues.

Effect of deletion of a trichothecene toxin regulatory gene on the secondary metabolism transcriptome of the saprotrophic fungus Trichoderma arundinaceum

Trichothecenes are terpenoid toxins produced by multiple fungal species with diverse lifestyles. In these fungi, the trichothecene biosynthetic gene (tri) cluster includes a gene encoding a Cys2His2 Zn-finger protein (TRI6). Analyses of plant pathogenic Fusarium species indicate that tri6 regulates tri gene expression. Here, we analyzed TRI6 function in the saprotrophic fungus Trichoderma arundinaceum, which produces the antimicrobial trichothecene harzianum A (HA). Deletion of the TRI6-encoding gene, tri6, blocked HA production and reduced expression of tri genes, and mevalonate biosynthetic genes required for synthesis of farnesyl diphosphate (FPP), the primary metabolite that feeds into trichothecene biosynthesis. In contrast, tri6 deletion did not affect expression of ergosterol biosynthetic genes required for synthesis of ergosterol from FPP, but did increase ergosterol production, perhaps because increased levels of FPP were available for ergosterol synthesis in the absence of trichothecene production. RNA-seq analyses indicated that genes in 10 of 49 secondary metabolite (SM) biosynthetic gene clusters in T. arundinaceum exhibited increased expression and five exhibited reduced expression in a tri6 deletion mutant (Δtri6). Despite the metabolic and transcriptional changes, Δtri6 mutants were not reduced in their ability to inhibit growth of fungal plant pathogens. Our results indicate that T. arundinaceum TRI6 regulates expression of both tri and mevalonate pathway genes. It remains to be determined whether the effects of tri6 deletion on expression of other SM clusters resulted because TRI6 can bind to promoter regions of cluster genes or because trichothecene production affects other SM pathways.

A highly efficient electrophoretic method for discrimination between two Neoscytalidium species using a specific fungal internal transcribed spacer (ITS) fragment.

Neoscytalidium (or N.) dimidiatum and N. novaehollandiae are two aggressive plant pathogenic species that affect several agricultural crops. Early detection and identification of these fungi are of critical importance to bring about the effective minimization to the threat they pose to the infected plants. Herein, two species of Neoscytalidium were rapidly discriminated by utilizing the rRNA internal transcribed (ITS4-5.8S-ITS5) PCR primers. A total of 100 isolates of Neoscytalidium species, which were isolated from Iraqi canker-infected fig trees, were included in this study. Two discrete electrophoretic PCR bands were observed in Neoscytalidium isolates-A-variants were about 546bp, while B-variants were about 993bp in length. The comprehensive phylogenetic analysis of both DNA variants revealed that A-variants resided between N. novaehollandiae and N. hyalinum, while B-variants were closely related to N. dimidiatum. Furthermore, the highly specific re-constructed tree of both electrophoretic variants demonstrated that B-variants share a high similarity with N. novaehollandiae. Additionally, the secondary structures for both variants were predicted computationally to reveal the structural patterns that each variant follows. In conclusion, a small rRNA locus comprising 22 nucleotides that differs in the two variants is potentially responsible for this species-specific classification. The main divergence in the amplified loci led to the classification of these fungal variants into two main species, namely N. dimidiatum and N. novaehollandiae, demonstrating that the amplification by ITS4-ITS5 rRNA fragment is a beneficial strategy that can be employed for the assessment of Neoscytalidium diversity in the natural ecosystems.

Contrasting distribution patterns between aquatic and terrestrial Phytophthora species along a climatic gradient are linked to functional traits

Diversity of microbial organisms is linked to global climatic gradients. The genus Phytophthora includes both aquatic and terrestrial plant pathogenic species that display a large variation of functional traits. The extent to which the physical environment (water or soil) modulates the interaction of microorganisms with climate is unknown. Here, we explored the main environmental drivers of diversity and functional trait composition of Phytophthora communities. Communities were obtained by a novel metabarcoding setup based on PacBio sequencing of river filtrates in 96 river sites along a geographical gradient. Species were classified as terrestrial or aquatic based on their phylogenetic clade. Overall, terrestrial and aquatic species showed contrasting patterns of diversity. For terrestrial species, precipitation was a stronger driver than temperature, and diversity and functional diversity decreased with decreasing temperature and precipitation. In cold and dry areas, the dominant species formed resistant structures and had a low optimum temperature. By contrast, for aquatic species, temperature and water chemistry were the strongest drivers, and diversity increased with decreasing temperature and precipitation. Within the same area, environmental filtering affected terrestrial species more strongly than aquatic species (20% versus 3% of the studied communities, respectively). Our results highlight the importance of functional traits and the physical environment in which microorganisms develop their life cycle when predicting their distribution under changing climatic conditions. Temperature and rainfall may be buffered differently by water and soil, and thus pose contrasting constrains to microbial assemblies.

Specific enzyme functionalities of Fusarium oxysporum compared to host plants

The genus Fusarium contains some of the most studied and important species of plant pathogens that economically affect world agriculture and horticulture. Fusarium spp. are ubiquitous fungi widely distributed in soil, plants as well as in different organic substrates and are also considered as opportunistic human pathogens. The identification of specific enzymes essential to the metabolism of these fungi is expected to provide molecular targets to control the diseases they induce to their hosts. Through applications of traditional techniques of sequence homology comparison by similarity search and Markov modeling, this report describes the characterization of enzymatic functionalities associated to protein targets that could be considered for the control of root rots induced by Fusarium oxysporum. From the analysis of 318 F. graminearum enzymes, we retrieved 30 enzymes that are specific of F. oxysporum compared to 15 species of host plants. By comparing these 30 specific enzymes of F. oxysporum with the genome of Arabidopsis thaliana, Brassica rapa, Glycine max, Jatropha curcas and Ricinus communis, we found 7 key specific enzymes whose inhibition is expected to affect significantly the development of the fungus and 5 specific enzymes that were considered here to be secondary because they are inserted in pathways with alternative routes.

A rapid, sensitive and field-deployable isothermal assay for the detection of Verticillium alfalfae

The genus Verticillium contains 10 plant pathogenic species that are responsible for billions of dollars of crop losses annually on more than 400 plant species worldwide. On alfalfa, Verticillium alfalfae causes Verticillium wilt, a disease with the potential to reduce yields of susceptible cultivars by the second harvest year and to limit the productive stand life to less than 3 years. Susceptible alfalfa cultivars exhibit disease symptoms that include chlorotic V-shaped lesions at the leaf tip that cause entire leaflets to become bleached and twisted, leading to defoliation. Stems often remain green after all leaflets have desiccated, and discolouration, stunting and wilting of shoots is visible. Internally, the taproot can show yellow to brown vascular discolouration. International quarantines sometimes prohibit the entry of alfalfa samples contaminated with the Verticillium pathogen. In this investigation, we developed a recombinase polymerase amplification (RPA) assay coupled with a lateral flow device for the specific and sensitive detection of V. alfalfae. The primers targeted the translation elongation factor 1α (TEF-1α) gene and were designed with mismatches to increase the specificity of the assay. This assay detected as little as 800 fg of DNA from all tested isolates of V. alfalfae. Moreover, non-specific amplification of closely related V. nonalfalfae or other Verticillium species was not observed. Detection of V. alfalfae in inoculated alfalfa cultivars with high or low resistance further demonstrated the specificity and sensitivity of the diagnostic assay. Additionally, RPA detected V. alfalfae from inoculated plants using a crude DNA extraction method. The assay is easy to use and will allow growers, diagnostic labs and regulatory agencies to determine if V. alfalfae is present in alfalfa products.

Taxonomic diversity of fungi deposited from the atmosphere

Fungi release spores into the global atmosphere. The emitted spores are deposited to the surface of the Earth by sedimentation (dry deposition) and precipitation (wet deposition), and therefore contribute to the global cycling of substances. However, knowledge is scarce regarding the diversities of fungi deposited from the atmosphere. Here, an automatic dry and wet deposition sampler and high-throughput sequencing plus quantitative PCR were used to observe taxonomic diversities and flux densities of atmospheric fungal deposition. Taxon-specific fungal deposition velocities and aerodynamic diameters (da) were determined using a collocated cascade impactor for volumetric, particle-size-resolved air sampling. Large multicellular spore-producing dothideomycetes (da ≥ 10.0 μm) were predominant in dry deposition, with a mean velocity of 0.80 cm s–1 for all fungal taxa combined. Higher taxonomic richness was observed in fungal assemblages in wet deposition than in dry deposition, suggesting the presence of fungal taxa that are deposited only in wet form. In wet deposition, agaricomycetes, including mushroom-forming fungi, and sordariomycetes, including plant pathogenic species, were enriched, indicating that such fungal spores serve as nuclei in clouds, and/or are discharged preferentially during precipitation. Moreover, this study confirmed that fungal assemblage memberships and structures were significantly different between dry and wet deposition (P-test, p < 0.001). Overall, these findings suggest taxon-specific involvement of fungi in precipitation, and provide important insights into potential links between environmental changes that can disturb regional microbial communities (e.g., deforestation) and changes in precipitation patterns that might be mediated by changes in microbial communities in the atmosphere.