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

The role of genomic islands in virulence of Pectobacterium carotovorum subspecies brasiliensis on potatoes

Pectobacterium carotovorum subsp. carotovorum is primarily responsible for soft rotting of potato tubers, although several strains have been shown to cause blackleg of potato stems. In contrast, P. atrosepticum is best known as a seedborne pathogen that causes blackleg. It is also responsible for soft rotting of tubers. Given the taxonomic distance between these two species and that only some strains of P. carotovorum subsp. carotovorum are able to cause blackleg, it was hypothesised that the capacity of P. carotovorum subsp. carotovorum to invade potato stems and elicit blackleg may have evolved through independent acquisition of genomic islands (GIs). GIs are large chromosomal regions in bacteria that are acquired by horizontal gene transfer and often encode virulence factors. To address this hypothesis, the genomes of P. carotovorum subsp. carotovorum strains associated with blackleg disease of potatoes were compared to those of non-blackleg causing strains, to identify GIs in blackleg causing strains and novel virulence factors encoded on these islands. First, the identity of a highly aggressive P. carotovorum subsp. carotovorum strain ICMP19477 and other New Zealand isolates known to cause blackleg, was re-assessed using molecular and phylogenetic assays. These assays identified the isolates as P. carotovorum subsp. brasiliensis. Comparative genomics using the genome sequence obtained for ICMP19477 and three other Pectobacterium in this study, as well as the genomes of 10 soft rot erwiniae (SRE) obtained from public databases, identified a total of 69 genomic islands and 10 gene islets in the genome of ICMP19477. Many of these islands and islets harboured genes predicted to be associated with the virulence of this pathogen (e.g. genes encoding putative plant cell wall degrading enzymes, phytotoxins, secretion systems, sugar utilisation, etc.), yet only one was present exclusively in the P. atrosepticum and P. carotovorum subsp. brasiliensis isolates historically known to cause blackleg. This islet (sim gene islet) encodes a sugar:phosphotransferase system, which is important in the pathogenicity of vascular plant pathogens such as Erwinia amylovora. The majority of other GIs and islets carrying putative virulence genes were either not present in the genomes of all blackleg causing strains or were present in strains that did not cause blackleg (e.g. GI PbN1_GI24, which encoded a putative non-ribosomal peptide with similarity to syringomycin). To confirm the role of several putative virulence factors in virulence of P. carotovorum subsp. brasiliensis ICMP19477, knockout mutants were constructed by allelic exchange mutagenesis. Mutations in the non-ribosomal peptide synthetase cluster of P. carotovorum subsp. brasiliensis (on PbN1_GI24) did not result in significant differences in virulence of the wild type and the mutant in pathogenicity assays on potato. In contrast, single crossover mutants in the islets encoding the sugar:phosphotransferase system and phenolic acid decarboxylase significantly reduced virulence of the pathogen. Complementation studies still need to be conducted to prove their role in disease. In summary, this study has provided new insights into the impact of GIs on the virulence of P. carotovorum subsp. braisliensis and also on the mechanisms by which SRE cause diseases. These data suggest that the accumulation of multiple virulence factors on these elements might play a larger role in virulence of Pectobacterium than the acquisition of a specific gene or gene cluster

Draft genome sequence for ICMP 5702, the type strain of Pectobacterium carotovorum subsp. carotovorum that causes soft rot disease on potato

Pectobacterium species are economically important bacteria that cause soft rotting of potato tubers in the field and in storage. Here, we report the draft genome sequence of the type strain for P. carotovorum subsp. carotovorum, ICMP 5702 (ATCC 15713). The genome sequence of ICMP 5702 will provide an important reference for future phylogenomic and taxonomic studies of the phytopathogenic Enterobacteriaceae

The effect of vineyard management practices on the arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) have been shown to have potentially significant applications for sustainable agricultural ecosystems including vineyards. The increased diversity of AMF has been indicated to be beneficial and increase resilience to environmental changes. In order to effectively utilise AMF communities and their benefits in vineyard ecosystems, a better understanding of how management practices influence AMF diversity and composition is needed. Moreover, little is known as to whether AMF communities found in organically managed vineyards are distinct from those found under conventionally managed vineyards. Therefore, vineyards were surveyed across the Marlborough region in order to identify the AMF communities colonising the roots of different rootstocks grafted with Sauvignon Blanc and Pinot Noir varieties in both conventional and organic systems. The AMF communities were identified based on spores isolated from trap culture that were set up with the collected grapevine roots, and by next-generation sequencing (Illumina Miseq). The identified AMF species/genera belonged mainly to Glomeraceae and Claroideoglomeraceae followed by Diversisporaceae, Paraglomeraceae, Archaeosporaceae and Gigasporaceae. The results revealed a significant (P < 0.05) difference in AMF community diversity and composition between organic and conventional vineyards as well as a significant interaction between rootstock and the management practice. This result indicated that some rootstocks might be more suited to organic systems due to the AMF communities they support under this management practice. This finding could provide an increased benefit under organic systems supporting higher biodiversity

First report of blackleg and soft rot of potato caused by Pectobacterium carotovorum

Blackleg and stem rot of potato occur sporadically in New Zealand, causing economic damage under optimal temperature and humidity conditions for disease development. Both Pectobacterium atrosepticum (Pba) and P. carotovorum subsp. carotovorum (Pcc) have previously been isolated from potato tubers with soft rot symptoms in New Zealand (Crowhurst & Wright, 1998) whereas only Pba has been shown to cause blackleg. A collection of 89 enterobacteria were recently isolated from potato tubers from commercial crops in the Auckland, Waikato, Manawatu-Wanganui and Canterbury regions of New Zealand. The majority were initially assigned as Pcc by their growth at 37°C, carbon utilisation profiles and restriction fragment length polymorphisms (Pitman et al., 2008). These isolates were mostly unable to cause blackleg symptoms, although several were shown to be highly aggressive upon stem infection

Draft genome sequences of three Pectobacterium strains causing blackleg of potato: P. carotovorum subsp. brasiliensis ICMP 19477, P. atrosepticum ICMP 1526, and P. carotovorum subsp. carotovorum UGC32

Blackleg is a disease caused by several species of Pectobacterium that results in losses to potato crops worldwide. Here, we report the draft genomes of three taxonomically and geographically distinct blackleg-causing strains of Pectobacterium: P. carotovorum subsp. brasiliensis ICMP 19477, P. atrosepticum ICMP 1526, and P. carotovorum subsp. carotovorum UGC32. Comparison of these genomes will support the identification of common traits associated with their capacity to cause blackleg

Pectobacterium atrosepticum and Pectobacterium carotovorum harbor distinct, independently acquired integrative and conjugative elements encoding coronafacic acid that enhance virulence on potato stems

Integrative and conjugative elements (ICEs) play a central role in the evolution of bacterial virulence, their transmission between bacteria often leading to the acquisition of virulence factors that alter host range or aggressiveness. Much is known about the functions of the virulence determinants that ICEs harbor, but little is understood about the cryptic effects of ICEs on their host cell. In this study, the importance of horizontally acquired island 2 (HAI2), an ICE in the genome of Pectobacterium atrosepticum SCRI1043, was studied using a strain in which the entire ICE had been removed by CRISPR-Cas-mediated genome editing. HAI2 encodes coronafacic acid, a virulence factor that enhances blackleg disease of potato stems caused by P. atrosepticum SCRI1043. As expected, deletion of HAI2 resulted in reduced blackleg symptoms in potato stems. A subsequent screen for HAI2-related ICEs in other strains of the Pectobacterium genus revealed their ubiquitous nature in P. atrosepticum. Yet, HAI2-related ICEs were only detected in the genomes of a few P. carotovorum strains. These strains were notable as blackleg causing strains belonging to two different subspecies of P. carotovorum. Sequence analysis of the ICEs in different strains of both P. atrosepticum and P. carotovorum confirmed that they were diverse and were present in different locations on the genomes of their bacterial host, suggesting that the cfa cluster was probably acquired independently on a number of occasions via chromosomal insertion of related ICEs. Excision assays also demonstrated that the ICEs in both P. atrosepticum and P. carotovorum are mobilized from the host chromosome. Thus, the future spread of these ICEs via lateral gene transfer might contribute to an increase in the prevalence of blackleg-causing strains of P. carotovorum