The effect of dual inoculation (Seimatosporium species with/without GTD fungi) on lesion length (symptom expression) in Sauvignon Blanc vines

In a survey of the endophytic fungal diversity associated with grapevines symptomatic or asymptomatic for grapevine trunk-diseases (GTDs) carried out in Marlborough, New Zealand in 2018, several fungal pathogens were isolated. Among these, members of the Botryosphaeriaceae family, Neofusicoccum parvum (from symptomatic vines) and Diplodia seriata (from both symptomatic and asymptomatic vines) were recovered. These pathogens are considered latent and virulent GTDs. Additionally, two Seimatosporium species, S. vitis and S. lichenicola, were recovered for the first time associated with GTD fungi in New Zealand vines. Both species were isolated from symptomatic and asymptomatic tissues, but their role as pathogens and interaction within GTD complexes is unclear. This study investigated the interaction between these Seimatosporium spp. and N. parvum or D. seriata in the GTD complex and the effect on symptom expression. The outcomes of in planta dual inoculation experiments between Seimatosporium spp. and N. parvum or D. seriata isolated from the same wood cankers were evaluated. Detached Sauvignon blanc grapevine green shoots and two-year-old woody stems of potted grapevines were wounded and co-inoculated with mycelial colonised agar discs of S. vitis or S. lichenicola and N. parvum or D. seriata. Controls consisted of each fungal species inoculated alone. After 2 weeks for detached shoots and 4 months for attached shoots, lesion length and colonisation distance by re-isolation were assessed. In both assays, there were differences in the lesion lengths and pathogen movement for co inoculation of both Seimatosporium spp. with N. parvum. In contrast, co-inoculation of either Seimatosporium spp. with D. seriata did not develop a lesion, although D. seriata were recovered at a distance of 5 cm upward and downward from the inoculation point. No lesions developed with D. seriata, S. vitis, or S. lichenicola inoculation alone. Our finding confirm that Seimatosporium spp. are involved in the GTD complex

Genetic diversity and virulence variability in Diplodia mutila isolates from symptomatic grapevines in New Zealand: Virulence and genetic diversity of Diplodia mutila

Genetic diversity and virulence variability of Diplodia mutila isolates recovered from grapevines in New Zealand were investigated. The universally primed PCR (UP-PCR) and vegetative compatibility group (VCG) methods were used to investigate the genetic diversity. Pathogenicity tests with ‘Sauvignon Blanc’ detached shoots and potted vines were used to determine the virulence diversity. UP-PCR analysis determined eight genetic groups of D. mutila with 70% of the population within one group. Phylogenetic analysis also determined that New Zealand isolates were more closely related to Australian isolates than Californian isolates. Vegetative compatibility grouping analysis placed the isolates into three VCG groups, with 57% of isolates belonging to all three VCGs. Vegetative compatibility reactions were observed among isolates, but this was not correlated with the genetic clustering. Virulence assays proved that all isolates tested were pathogenic on grapevine stems. Differences in necrotic lesions lengths caused by D. mutila isolates were identified, indicating different virulence levels among isolates, however, no relationship was found between the genetic groups and the virulence. The results of the study indicated movement of D. mutila isolates between nurseries, vineyards, and other sources in New Zealand. This information will inform control strategies to limit the further spread of this pathogen into vineyards in the same region or new regions

Development of an isolate-specific marker for tracking Phaeomoniella chlamydospora infection in grapevines

Petri disease causes decline of grapevines worldwide. The grapevine endophyte Phaeomoniella chlamydospora is the most important fungal pathogen associated with this disease. Epidemiological studies of this pathogen have been hampered by its common occurrence in the internal tissue of apparently healthy vines. Development of a molecular marker for a single strain would overcome this limitation and aid experiments designed to answer key questions about the biology of this pathogen. Genetic variation analysis of New Zealand and Italian strains of P. chlamydospora detected a potential molecular marker in New Zealand isolate A21. Characterization of the 1010 bp marker band showed that it had 50% identity to moxY, a gene involved in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. Sequencing of the region flanking the 1010 bp product revealed a single nucleotide polymorphism in the 3' border of the marker band. Primers were designed to amplify a 488 bp fragment encompassing this polymorphic site and cleavage of this product with the restriction enzyme BsrI produced three bands only in isolate A21 and two bands in all other isolates tested. The sensitivity of the PCR-RFLP protocol was increased with a nested PCR approach and the protocol optimized for soil and wood samples. When the nested PCR/RFLP procedure was used to determine the persistence of viable and nonviable spores in soil, the results showed that nonviable spores were undetected after 8 wk whereas viable spores still could be detected at 17 wk

Culturome versus DNA metabarcoding: Diversity of grapevine endophytic mycobiome in old and young vines of different health status in New Zealand

The grapevine harbours a diverse community of fungi in the woody trunk tissue, termed the “endophytic mycota”. These communities can have a profound effect on the vine’s physiology, health, growth, and ability to adapt to stress. Some of these include pathogenic fungi as the causal agents of grapevine trunk disease (GTD), with many considered latent pathogens. For GTD, understanding the factors affecting latency is still limited. This study aimed to compare the fungal endophyte community in young and old Sauvignon blanc vines, both symptomatic and asymptomatic for GTD, using culture-dependent and culture-independent approaches. Nine vineyards were sampled, with 60 mature vines (>10 years old) and 30 young vines (<9 years old) sampled. Each age group consisted of equal numbers of apparently healthy and symptomatic vines. Trunk cores were taken from each vine using a sterilised 4-mm drill bit after removing the bark with a knife. Fungal communities were characterized by isolation and metabarcoding of the ITS1 region. For the culturome, a collection of 2116 endophytic fungi were recovered, representing 42 fungal genera. Trunk microbiota was dominated by species of the genera Alternaria, Aureobasidium, Diplodia,Epicoccum, Phaeomoniella, Eutypa, Botrytis, Cladosporium, and Diaporthe. Differences in the taxa recovered into culture were observed between vines of different ages, and symptomology. In the metabarcoding approach, 1892 OTUs were obtained. The same fungal genera were identified as the most abundant using metabarcoding. Alpha diversity analysis revealed that greater diversity was detected in old compared to young vines and in asymptomatic compared to symptomatic trunks. Beta diversity analysis demonstrated significant differentiation in the fungal communities structure for both age and health status. This study has produced new baseline information on Sauvignon blanc endophytic mycota and further work will determine the impact of these microbial communities on the latency of GTDs

Can the microbiome drive the suppression of grapevine trunk diseases?

Grapevine trunk diseases (GTDs), caused by several fungal species, are among the most destructive grapevine diseases in New Zealand and other grape-growing countries. The control of the diseases is problematic, and there is currently no approved fungicide for their eradication. This has necessitated seeking alternative strategies, including a sustainable biological control approach, to manage the diseases. Therefore, this study aimed to identify taxa in the grapevine microbiome that contribute to plant health. In some New Zealand vineyards, observations have revealed vines that remain healthy within a background of trunk diseases. These grapevines were termed ‘disease-escape’ to represent their apparent health under heavy disease pressure. Recent research on the grapevine microbiome has shown that microorganisms from these ‘disease-escape’ plants could contribute to disease suppression. Putative disease escape vines were identified in vineyards in two grape-growing regions in New Zealand: Hawke’s Bay and Canterbury. The vines were selected based on their presence in a diseased area, maturity, and absence of trunk disease symptoms. Trunk core samples were taken from the disease-escape vines and neighbouring symptomatic vines. Subsequently, the samples’ total fungal and bacterial communities were identified and compared using culture-independent DNA metabarcoding and culture-dependent approaches. After analysing the metabarcoding and culturing results, microbial taxa that were differentially more abundant in disease-escape grapevines and the ones that correlated negatively with GTD pathogens were identified. The next stage of the study is to design a synthetic community using members of the taxa of interest from the disease-escape grapevines. This SynCom will be introduced into young grapevines and monitored for their ability to suppress the development and severity of GTDs. The research results will provide information on the roles (if any) that the grapevine trunk’s microbiome plays in suppressing GTDs

Influence of blueberry tissue type, wounding and cultivar on susceptibility to infection by Neofusicoccum species

Aim: Botryosphaeriaceae causing stem blight and dieback of blueberry are important pathogens limiting economic production worldwide. This study investigated the pathogenicity and relative virulence of isolates from the Neofusicoccum species commonly associated with blueberries in New Zealand on different tissues and cultivars of blueberries. Methods and Results: Both wounded and non- wounded fruit and flower buds and wounded attached soft green and hard green shoots were susceptible to infection by conidia of Neofusicoccum australe, Neofusicoccum parvum and Neofusicoccum ribis. N. ribis was generally most virulent, followed by N. parvum and then N. australe. Inoculation of potting mixture with N. australe or N. ribis conidia showed that potting mixtures were not a source of inoculum for infection of blueberry roots. Wounded and non- wounded leaf buds, fruit and wounded soft green shoots and hard green shoots of the different cultivars tested were susceptible to infection by N. parvum and N. ribis. Whilst the fruit of all cultivars were similarly infected, infection incidence in inoculated leaf buds was lowest in “Blue Bayou” and “Ocean Blue”. Cultivar susceptibility differed when tested on soft green shoots compared with hard green shoots, with shortest lesions developed on “Maru” on soft green shoots, and “Centra Blue” and “Ocean Blue” on hard green shoots. Conclusions: All tested above- ground blueberry tissues, including non- wounded tissue, were susceptible to Neofusicoccum spp. All the cultivars assessed were susceptible to infection, although they varied in their relative susceptibility depending on the tissue assessed. Significance and impact of the study: The potential for non- wounded tissue to become infected indicate that fungicides may need to be applied to protect all tissue, not just wounds

Determining the presence of host specific toxin genes, ToxA and ToxB, in New Zealand Pyrenophora tritici-repentis isolates, and susceptibility of wheat cultivars

Tan spot, caused by Pyrenophora tritici-repentis (Ptr), is an important disease of wheat worldwide, and an emerging issue in New Zealand. The pathogen produces host-specific toxins which interact with the wheat host sensitivity loci. Identification of the prevalence of the toxin encoding genes in the local population, and the susceptibility of commonly grown wheat cultivars to Ptr will aid selection of wheat cultivars to reduce disease risk. Twelve single spore isolates collected from wheat-growing areas of the South Island of New Zealand representing the P. tritici-repentis population were characterised for the Ptr ToxA and ToxB genes, ToxA and ToxB, respectively, using two gene specific primers. The susceptibility of 10 wheat cultivars to P. tritici-repentis was determined in a glasshouse experiment by inoculating young plants with a mixed-isolate spore inoculum. All 12 New Zealand P. tritici-repentis isolates were positive for the ToxA gene but none were positive for the ToxB gene. Tan spot lesions developed on all inoculated 10 wheat cultivars, with cultivars ‘Empress’ and ‘Duchess’ being the least susceptible and ‘Discovery’, ‘Reliance’ and ‘Saracen’ the most susceptible cultivars to infection by the mixed-isolate spore inoculum used. The results indicated that the cultivars ‘Empress’ and ‘Duchess’ may possess a level of tolerance to P. tritici-repentis and would, therefore, be recommended for cultivation in regions with high tan spot incidence

Grapevines escaping trunk diseases in New Zealand vineyards have a distinct microbiome structure

Grapevine trunk diseases (GTDs) are a substantial challenge to viticulture, especially with a lack of available control measures. The lack of approved fungicides necessitates the exploration of alternative controls. One promising approach is the investigation of disease escape plants, which remain healthy under high disease pressure, likely due to their microbiome function. This study explored the microbiome of grapevines with the disease escape phenotype. DNA metabarcoding of the ribosomal internal transcribed spacer 1 (ITS1) and 16S ribosomal RNA gene was applied to trunk tissues of GTD escape and adjacent diseased vines. Our findings showed that the GTD escape vines had a significantly different microbiome compared with diseased vines. The GTD escape vines consistently harbored a higher relative abundance of the bacterial taxa Pseudomonas and Hymenobacter. Among fungi, Aureobasidium and Rhodotorula were differentially associated with GTD escape vines, while the GTD pathogen, Eutypa, was associated with the diseased vines. This is the first report of the link between the GTD escape phenotype and the grapevine microbiome

Similar strains of Burkholderia spp. nodulate the South African invasive legume Dipogon lignosus in New Zealand and Australian soils

Brazil and South Africa are centres of diversity of Burkholderia ssp. that nodulate legumes (Gyaneschwar et al, 2011; Beukes et al. 2013). The nod gene sequence of Burkholderia spp, capable of nodulating South Africa plants are clearly separated from those of Burkholderia spp. shown to nodulate South American plants. Where tested, the South African strains did not nodulate South American plants nodulated by Burkholderia spp. (Gyaneschwar et al. 2011)

AMF community diversity promotes grapevine growth parameters under high black foot disease pressure

Black foot disease is one of the main grapevine root diseases observed worldwide and is especially problematic in New Zealand. Arbuscular mycorrhizal fungi (AMF) have been shown to reduce infection and mitigate the effect of black foot disease on grapevine rootstocks. In contrast to prior studies, which have limited their focus to the effect of one, two or a combination of only a small number of AMF species, this study used whole AMF communities identified from 101-14, 5C and Schwarzmann rootstocks sampled from New Zealand vineyards. The effect of AMF on black foot disease was investigated in a ‘home’ and ‘away’ experiment using three commercial grapevine rootstocks. The study produced some evidence that AMF treatments lowered disease incidence at 5 cm and disease severity in vines by 40% to 50% compared to the vines inoculated with the pathogen only. This work also showed that the presence of high disease incidence may have limited the potential disease protective effect of AMF community. However, despite the high disease incidence and severity, AMF inoculation increased vine growth parameters by 60% to 80% compared to the vines inoculated with the pathogen only. This study is the first to provide an understanding on how young grapevine rootstocks inoculated with their ‘home’ and ‘away’ AMF communities would respond to challenge with a black foot pathogen species mixture. Further research is required to understand the mechanistic effect of AMF colonization on the increase of grapevine growth parameters under high black foot disease pressure