Phytotoxic Metabolites from Three Neofusicoccum Species Causal Agents of Botryosphaeria Dieback in Australia, Luteopyroxin, Neoanthraquinone, and Luteoxepinone, a Disubstituted Furo-α-pyrone, a Hexasubstituted Anthraquinone, and a Trisubstituted Oxepi-2-one from Neofusicoccum luteum.

Different phytotoxic metabolites were isolated from the organic extract of Neofusicoccum luteum, Neofusicoccum australe, and Neofusicoccum parvum, causal agents of Botryosphaeria dieback in Australia. N. luteum produced a new disubstituted furo-α-pyrone, a hexasubstituted anthraquinone, and a trisubstituted oxepi-2(7H)-one, luteopyroxin (4), neoanthraquinone (5), and luteoxepinone (7), respectively, together with the known (±)-nigrosporione (6), tyrosol (8), (R)-(-)-mellein (1), and (3R,4S)-(-)- and (3R,4R)-(-)-4-hydroxymellein (2 and 3). The three melleins and tyrosol were also produced by N. parvum, while N. australe produced (R)-(-)-mellein (1), neoanthraquinone (5), tyrosol (8), and p-cresol (9). Luteopryoxin (4), neoanthraquinone (5), and luteoxepinone (7) were characterized by analyses of physical data, essentially one- and two-dimensional nuclear magnetic resonance and high-resolution electrospray ionization mass spectrometry. The relative and absolute configurations of luteopyroxin (4) were determined by nuclear Overhauser effect spectroscopy and experimental and calculated electronic circular dichroism data. When assayed on grapevine leaves, neoanthraquinone (5) showed the highest toxic effect, causing severe shriveling and withering. Luteopyroxin (4), nigrosporione (6), and luteoxepinone (7) also showed different degrees of toxicity, while p-cresol (9) displayed low phytotoxicity.

Syringopeptin Contributes to the Virulence of Pseudomonas fuscovaginae, Based on sypA Biosynthesis Mutant Analysis.

Pseudomonas fuscovaginae, first reported from Japan in 1976, is now present in many agroecological regions around the world; it causes sheath brown rot of rice and is reported as a pathogen of a broad range of hosts. The pathogen can infect rice plants at all stages of growth and is known to cause significant losses due to grain discoloration, poor spike emergence and panicle sterility. Limited information is available on the virulence and mechanisms of pathogenicity for P. fuscovaginae. To address this, an analysis of genomes was conducted, which identified the presence of a gene showing homology to one of the genes contributing to syringopeptin synthetase (sypA) of P. syringae pv. syringae. To study the potential role of this gene in the virulence and pathogenicity of P. fuscovaginae, a site-specific mutation was created. Following inoculation of seeds and plantlets of rice and wheat with P. fuscovaginae wild types and their respective mutants, we demonstrated that the mutation significantly reduced virulence. This was evident on rice and wheat inoculated with mutants causing a significantly higher number of roots, length of roots and seedling height compared with their respective wild types. Characteristic disease symptoms of necrotic lesions were significantly less in rice seedlings infected with bacterial suspensions of mutants indicating a reduction in virulence. Chromatography analysis of bacterial exudates showed suppression of synthesis of metabolites analogous to syringopeptin in the mutants. These data demonstrate that the protein encoded by this sypA homolog gene is a major virulence determinant of P. fuscovaginae.

Occurrence and diversity of entomopathogenic fungi (Beauveria spp. and Metarhizium spp.) in Australian vineyard soils.

Entomopathogenic Ascomycetes: Hypocreales fungi occur worldwide in the soil; however, the abundance and distribution of these fungi in a vineyard environment is unknown. A survey of Australian vineyards was carried out in order to isolate and identify entomopathogenic fungi. A total of 240 soil samples were taken from eight vineyards in two states (New South Wales and Victoria). Insect baiting (using Tenebrio molitor) and soil dilution methods were used to isolate Beauveria spp. and Metarhizium spp. from all soil samples. Of the 240 soil samples, 60% contained either Beauveria spp. (26%) or Metarhizium spp. (33%). Species of Beauveria and Metarhizium were identified by sequencing the B locus nuclear intergenic region (Bloc) and elongation factor-1 alpha (EFT1) regions, respectively. Three Beauveria species (B. bassiana, B. australis and B. pseudobassiana) and six Metarhizium species (M. guizhouense, M. robertsii, M. brunneum, M. flavoviride var. pemphigi, M. pingshaense and M. majus) were identified. A new sister clade made up of six isolates was identified within B. australis. Two potentially new phylogenetic species (six isolates each) were found within the B. bassiana clade. This study revealed a diverse community of entomopathogenic fungi in sampled Australian vineyard soils.

Molecular Methods to Detect and Quantify Botryosphaeriaceae Inocula Associated With Grapevine Dieback in Australia.

Botryosphaeria dieback, caused by species of Botryosphaeriaceae, is an important grapevine trunk disease in Australia. Inocula produced by the pathogens are primarily dispersed by rain splash and wind and infect pruning wounds leading to cankers, dieback, and eventually death of vines. The objective of this study was to develop molecular tools to detect and quantify Botryosphaeriaceae inocula from the environment. These tools are essential for investigating spore dispersal patterns of Botryosphaeriaceae pathogens in Australian vineyards. DNA extraction protocols were evaluated and one modified protocol was found suitable for extracting Botryosphaeriaceae DNA from artificially and naturally inoculated Burkard volumetric spore sampler tapes. Multispecies primers and a hydrolysis probe for quantitative PCR (qPCR) were further developed to detect and quantify Botryosphaeriaceae inocula from environmental samples. Specificity tests showed that the multispecies primers were able to amplify the DNA of 10 Botryosphaeriaceae species (58 isolates) found in Australia while none of the 27 nontarget fungal species (90 isolates) tested were amplified. The qPCR assay was suitable for amplifying purified DNA, synthetic DNA fragments (gBlocks), and mixed DNA from spore trap tapes. The qPCR method developed in this study was shown to be rapid and sensitive in detecting Botryosphaeriaceae inocula from the environment using spore traps.

Pathogenicity of Diatrypaceous Fungi on Grapevines in Australia.

In addition to Eutypa lata, which causes Eutypa dieback, numerous other fungi in the Diatrypaceae family have been isolated from diseased grapevines (Vitis vinifera) and other woody hosts. Pathogenicity trials comprising 70 strains of diatrypaceous fungi representing nine species in six genera were conducted to determine whether these fungi, collected in Australia, were pathogenic to grapevines. When inoculated into wounded trunks of 'Cabernet Sauvignon', eight species, including E. lata, E. leptoplaca, Cryptovalsa ampelina, C. rabenhorstii, Eutypella citricola, E. microtheca, Diatrypella vulgaris, and a Diatrype sp. produced necrotic lesions significantly longer than on controls. In addition, all nine species (including a Cryptosphaeria sp.) were reisolated from the margins of developing lesions and at varying distances above and below the point of inoculation. Diatrypaceous fungi were frequently isolated from asymptomatic or otherwise healthy tissue several centimeters ahead of the disease margin. Methods to control diseases associated with diatrypaceous fungi must take into account their propensity to colonize woody tissues ahead of or in the absence of visible symptoms. Current recommendations for the management of Eutypa dieback using remedial surgery and pruning wound protection appear sufficient for the control of the other diatrypaceous fungi included in this study.

Biocontrol Potential of an Endophytic Pseudomonas poae Strain against the Grapevine Trunk Disease Pathogen Neofusicoccum luteum and Its Mechanism of Action.

Grapevine trunk diseases (GTDs) impact the sustainability of vineyards worldwide and management options are currently limited. Biological control agents (BCAs) may offer a viable alternative for disease control. With an aim to develop an effective biocontrol strategy against the GTD pathogen Neofusicoccum luteum, this study investigated the following: (1) the efficacy of the strains in suppressing the BD pathogen N. luteum in detached canes and potted vines; (2) the ability of a strain of Pseudomonas poae (BCA17) to colonize and persist within grapevine tissues; and (3) the mode of action of BCA17 to antagonize N. luteum. Co-inoculations of the antagonistic bacterial strains with N. luteum revealed that one strain of P. poae (BCA17) suppressed infection by 100% and 80% in detached canes and potted vines, respectively. Stem inoculations of a laboratory-generated rifampicin-resistant strain of BCA17 in potted vines (cv. Shiraz) indicated the bacterial strain could colonize and persist in the grapevine tissues, potentially providing some protection against GTDs for up to 6 months. The bioactive diffusible compounds secreted by BCA17 significantly reduced the spore germination and fungal biomass of N. luteum and the other representative GTD pathogens. Complementary analysis via MALDI-TOF revealed the presence of an unknown cyclic lipopeptide in the bioactive diffusible compounds, which was absent in a non-antagonistic strain of P. poae (JMN13), suggesting this novel lipopeptide may be responsible for the biocontrol activity of the BCA17. Our study provided evidence that P. poae BCA17 is a potential BCA to combat N. luteum, with a potential novel mode of action.

Molecular detection and identification of Diatrypaceous airborne spores in Australian vineyards revealed high species diversity between regions.

The grapevine trunk disease, Eutypa dieback (ED), causes significant vine decline and yield reduction. For many years, the fungus Eutypa lata was considered the main pathogen causing ED of grapevines in Australia. Recent studies showed other Diatrypaceous fungi were also associated with vines exhibiting dieback symptoms but there is limited information on how these fungal pathogens spread in vineyards. Thus, information on the spore dispersal patterns of Diatrypaceous fungi in different wine regions will assist in identifying high-risk infection periods in vineyards. Using more than 6800 DNA samples from airborne spores collected from eight wine regions in south-eastern Australia over 8 years using a Burkard spore trap, this study investigated the diversity and abundance of Diatrypaceous species, using multi-faceted molecular tools. A multi-target quantitative PCR (qPCR) assay successfully detected and quantified Diatrypaceous spores from 30% of the total samples with spore numbers and frequency of detection varying between regions and years. The high-resolution melting analysis (HRMA) coupled with DNA sequencing identified seven species, with E. lata being present in seven regions and the most prevalent species in the Adelaide Hills, Barossa Valley and McLaren Vale. Cryptovalsa ampelina and Diatrype stigma were the predominant species in the Clare Valley and Coonawarra, respectively while Eutypella citricola and Eu. microtheca dominated in the Hunter Valley and the Riverina regions. This study represents the first report of D. stigma and Cryptosphaeria multicontinentalis in Australian vineyards. This study further showed rainfall as a primary factor that triggers spore release, however, other weather factors that may influence the spore release in different climatic regions of Australia still requires further investigation.

Evaluation of Fungicides for the Management of Botryosphaeria Canker of Grapevines.

The family Botryosphaeriaceae comprises a number of species that are associated with the dieback disease of grapevine (Vitis vinifera), referred to as Botryosphaeria canker. To date, there are few effective agents available for the management of this disease. In this study, fungicides were evaluated for controlling the disease using a combination of in vitro tests and field trials. Twenty fungicides registered for use on other diseases in Australian viticulture were tested in vitro for their effect on mycelial growth of four species within the Botryosphaeriaceae. The concentrations of fungicide at which 50% of mycelial growth is inhibited (EC50 values) were significantly affected both by fungicide and isolate (P < 0.001). Differences in sensitivities of the four species to the fungicides were negligible (0.41 to 0.59 mg/liter). The most effective fungicides were fludioxonil, carbendazim, fluazinam, tebuconazole, flusilazole, penconazole, procymidone, iprodione, myclobutanil, and pyraclostrobin, for which EC50 values were <1.0 mg/liter. These fungicides were evaluated under field conditions, in addition to the pruning wound protectants Bacseal Super, Garrison, and ATCS tree wound dressing, as well as the biological control agent Vinevax. In field trials, carbendazim (Bavistin), fluazinam (Shirlan), tebuconazole (Folicur), Garrison, and ATCS tree wound dressing applied to freshly cut pruning wounds were the most effective and reduced infection by Diplodia seriata and D. mutila by 41 to 65%. These results suggest that the occurrence of Botryosphaeria canker on grapevines may be reduced via treatment of pruning wounds with selected fungicides as soon as possible after pruning.

Phytotoxic Metabolites Produced by Fungi Involved in Grapevine Trunk Diseases: Progress, Challenges, and Opportunities.

Grapevine trunk diseases (GTDs), caused by fungal pathogens, are a serious threat to vineyards worldwide, causing significant yield and economic loss. To date, curative methods are not available for GTDs, and the relationship between the pathogen and symptom expression is poorly understood. Several plant pathologists, molecular biologists, and chemists have been investigating different aspects of the pathogenicity, biochemistry, and chemical ecology of the fungal species involved in GTDs. Many studies have been conducted to investigate virulence factors, including the chemical characterization of phytotoxic metabolites (PMs) that assist fungi in invading and colonizing crops such as grapevines. Moreover, multidisciplinary studies on their role in pathogenicity, symptom development, and plant-pathogen interactions have also been carried out. The aim of the present review is to provide an illustrative overview of the biological and chemical characterization of PMs produced by fungi involved in Eutypa dieback, Esca complex, and Botryosphaeria dieback. Moreover, multidisciplinary investigations on host-pathogen interactions, including those using cutting-edge Omics techniques, will also be reviewed and discussed. Finally, challenges and opportunities in the role of PMs for reliable field diagnosis and control of GTDs in vineyards will also be explored.

Detection and Monitoring of Greeneria uvicola and Colletotrichum acutatum Development on Grapevines by Real-Time PCR.

Bitter rot (Greeneria uvicola) and ripe rot (Colletotrichum acutatum, syn. C. simmondsii) occur frequently in subtropical grape-growing regions of Australia, where they cause yield loss and bitter taints in wine. To further advance the epidemiological studies of G. uvicola and C. acutatum and contribute toward their effective management and control, a rapid and reliable species-specific real-time polymerase chain reaction (PCR) method was developed based on the polymorphic portion of the internal transcribed spacer region of the two fungi. It was found that, within 6 to 8 h postinoculation, the assay could detect as little as 20 fg of genomic DNA and 10 conidia for both species. Artificially and naturally infected grape inflorescences and mature berries were analyzed by both conventional plating methods and real-time PCR. Fungal presence was demonstrated on all plant material but development was observed only on mature berries. The results demonstrate that the real-time PCR technique is a highly specific, rapid, and sensitive method that can be used to detect and study the dynamics of G. uvicola and C. acutatum during different stages of infection and on different grape tissues.

Luteoethanones A and B, two phytotoxic 1-substituted ethanones produced by Neofusicoccum luteum, a causal agent of Botryosphaeria dieback on grapevine.

Two new phytotoxic 1-substituted ethanones, named luteoethanones A and B, were isolated from Neofusicoccum luteum, the causal agents of Botryosphaeria dieback in Australia. Luteoethanones A and B were characterized, by spectroscopic methods (essentially 1 D and 2 D NMR and HR ESIMS), as 1-(8-Methoxy-2,4-methyl-1-oxa-4-aza-spiro[2.5]octa-5,7-dien-6-yl)-ethanone and its 2-demethyl analogue. When assayed on detached grapevine leaves (Vitis vinifera cv. Shiraz) both the compounds showed phytotoxic activity.

Production of Phytotoxic Metabolites by Botryosphaeriaceae in Naturally Infected and Artificially Inoculated Grapevines.

Grapevine trunk diseases (GTDs) are considered a serious problem to viticulture worldwide. Several GTD fungal pathogens produce phytotoxic metabolites (PMs) that were hypothesized to migrate to the foliage where they cause distinct symptoms. The role of PMs in the expression of Botryosphaeria dieback (BD) symptoms in naturally infected and artificially inoculated wood using molecular and analytical chemistry techniques was investigated. Wood samples from field vines naturally infected with BD and one-year-old vines inoculated with Diplodia seriata, Spencermartinsia viticola and Dothiorella vidmadera were analysed by cultural isolations, quantitative PCR (qPCR) and targeted LC-MS/MS to detect three PMs: (R)-mellein, protocatechuic acid and spencertoxin. (R)-mellein was detected in symptomatic naturally infected wood and vines artificially inoculated with D. seriata but was absent in all non-symptomatic wood. The amount of (R)-mellein detected was correlated with the amount of pathogen DNA detected by qPCR. Protocatechuic acid and spencertoxin were absent in all inoculated wood samples. (R)-mellein may be produced by the pathogen during infection to break down the wood, however it was not translocated into other parts of the vine. The foliar symptoms previously reported in vineyards may be due to a combination of PMs produced and climatic and physiological factors that require further investigation.

Diversity Profiling of Grapevine Microbial Endosphere and Antagonistic Potential of Endophytic Pseudomonas Against Grapevine Trunk Diseases.

Grapevine trunk diseases (GTDs) are a serious problem of grapevines worldwide. The microbiota of the grapevine endosphere comprises prokaryotic and eukaryotic endophytes, which may form varied relationships with the host plant from symbiotic to pathogenic. To explore the interaction between grapevine endophytic bacteria and GTDs, the endomicrobiome associated with grapevine wood was characterized using next-generation Illumina sequencing. Wood samples were collected from grapevine trunks with and without external symptoms of GTD (cankers) from two vineyards in the Hunter Valley and Hilltops, NSW, Australia and metagenomic characterization of the endophytic community was conducted using the 16S rRNA gene (341F/806R) and ITS (1F/2R) sequences. Among the important GTD pathogens, Phaeomoniella, Phaeoacremonium, Diplodia and Cryptovalsa species were found to be abundant in both symptomatic and asymptomatic grapevines from both vineyards. Eutypa lata and Neofusicoccum parvum, two important GTD pathogens, were detected in low numbers in Hilltops and the Hunter Valley, respectively. Interestingly, Pseudomonas dominated the bacterial community in canker-free grapevine tissues in both locations, comprising 56-74% of the total bacterial population. In contrast, the Pseudomonas population in grapevines with cankers was significantly lower, representing 29 and 2% of the bacterial community in Hilltops and the Hunter Valley, respectively. The presence of Pseudomonas in healthy grapevine tissues indicates its ability to colonize and survive in the grapevine. The potential of Pseudomonas spp. as biocontrol agents against GTD pathogens was also explored. Dual culture tests with isolated fluorescent Pseudomonas against mycelial discs of nine Botryosphaeria dieback, three Eutypa dieback, and two Esca/Petri disease pathogens, revealed antagonistic activity for 10 Pseudomonas strains. These results suggest the potential of Pseudomonas species from grapevine wood to be used as biocontrol agents to manage certain GTD pathogens.

Draft Genome Sequences of Endophytic Pseudomonas spp. Isolated from Grapevine Tissue and Antagonistic to Grapevine Trunk Disease Pathogens.

Endophytic strains of Pseudomonas were isolated from grapevine tissues and exhibited antagonistic activity against several grapevine trunk disease pathogens. The draft genome sequences of the four strains revealed the presence of putative gene clusters that may impart biocontrol activity against plant pathogens.

Phytotoxic metabolites by nine species of Botryosphaeriaceae involved in grapevine dieback in Australia and identification of those produced by Diplodia mutila, Diplodia seriata, Neofusicoccum australe and Neofusicoccum luteum.

Botryosphaeria dieback is one of the main trunk diseases of grapevine caused by several species of Botryosphaeriaceae. Twenty-four fungal isolates representing the eight most widespread and most virulent Botryosphaeriaceae were tested for their ability to produce phytotoxic metabolites. The chromatographic profiles of their culture filtrates organic extracts showed the ability of all isolates to produce several and different metabolites. When tested on grapevine leaves and tomato cuttings the organic extracts phytotoxicity varied among isolates and species. To our knowledge, this is the first study on phytotoxic compounds produced by Botryosphaeriaceae species found in Australian vineyards. The phytotoxic metabolites produced by Diplodia seriata, Diplodia mutila, Neofusicoccum australe and, for the first time, by Neofusicoccum luteum were isolated and chemically identified essentially by spectroscopic methods.

Diploquinones A and B, Two New Phytotoxic Tetrasubstituted 1,4-Naphthoquinones from Diplodia mutila, a Causal Agent of Grapevine Trunk Disease.

Two new phytotoxic tetrasubstituted 1,4-naphthoquinones, named diploquinones A and B, were isolated together with vanillic acid from Diplodia mutila (DAR78993), a grapevine pathogen involved in Botryosphaeria dieback in Australia. Diploquinones A and B were characterized as 6,7-dihydroxy-2-methoxy-5-methylnaphthalene-1,4-dione and 3,5,7-trihydroxy-2-methoxynaphthalene-1,4-dione using spectroscopic methods (essentially 1D and 2D 1H and 13C NMR and HR ESIMS). The already known vanillic acid was isolated for the first time as fungal phytotoxin and as metabolite of D. mutila. The three compounds were assayed on detached grapevine leaves ( Vitis vinifera cv. Shiraz) at concentrations of 10-3 M and 2.5 × 10-3 M. Vanillic acid showed the highest phytotoxic effect on grapevine leaves irrespective of the tested concentration, while diploquinones A and B showed varying degrees of toxicity.

Isolation of Phytotoxic Phenols and Characterization of a New 5-Hydroxymethyl-2-isopropoxyphenol from Dothiorella vidmadera, a Causal Agent of Grapevine Trunk Disease.

Polyphenols were characterized from Dothiorella vidmadera (DAR78993), which was isolated from a grapevine in Australia. In total, six polyphenols were isolated including a new polyphenol characterized by a spectroscopic method (essentially NMR and HR ESIMS) as 5-hydroxymethyl-2-isopropoxyphenol. Tyrosol, benzene-1,2,4-triol, resorcinol, 3-(hydroxymethyl)phenol, and protocatechuic alcohol, the latter being the main metabolite, were also isolated. Although these are already known as naturally occurring compounds in microorganisms and plants, this is the first time they have been isolated from fungal organisms involved in grapevine trunk disease. When assayed on tomato seedlings, all the compounds show similar phytotoxic effects. However, when assayed on grapevine leaves (Vitis vinifera cv Shiraz), resorcinol was the most toxic compound, followed by protocatechuic alcohol and 5-hydroxymethyl-2-isopropoxyphenol.