Anish Shah

The variability of the rhizosphere microbial community has not been well-studied in avocado plantations at the field-scale. This research aimed to determine if the bacterial and fungal communities in the rhizosphere and/or the soil physicochemical properties from two commercial avocado orchards varied with different sampling designs (grid-based, longitudinal transect, and zigzag transect), and in turn if this changed the soil physicochemical properties driving the composition of the microbial communities. There were no differences in alpha diversity of bacteria or fungi based on sampling design in either orchard, and bacterial and fungal alpha diversity showed no evidence of spatial autocorrelation. Bacterial community composition in Orchard 1 varied with sampling design, whereas no differences were observed for bacterial community composition in Orchard 2 or for fungal community composition in either orchard. In each orchard, at least 50 % of the most abundant bacterial taxa were common between the sampling designs, however, less than 40 % the most abundant fungal taxa were common between the sampling designs. Canonical correspondence analysis indicated that the edaphic drivers of bacterial and fungal communities in Orchard 1 differed based on the sampling design. These results highlight the importance of field-scale sampling design for accurately characterising avocado rhizosphere bacterial and fungal communities particularly when such data will inform orchard management decisions. Soil sampling using a random, grid-based design is recommended as a simple and reliable method for monoculture fruit tree orchards.

Roots of tomato plants (Solanum lycopersicum) are vulnerable to soil-borne pathogenic fungi, bacteria and nematodes. Current control methods for these biotic stressors have limitations, necessitating the need for new eco-friendly alternatives. The rhizosphere microbiome is an effective natural barrier to invasion by soil-borne pathogens, and there is scope to harness this inherent capacity to improve the management of pathogens. This review examined molecular analyses of the taxonomic composition, abundance and function of the rhizosphere microbiome in healthy and diseased field-grown tomato plants for evidence of the role of the microbiome in disease suppression. The role of biological products in manipulating the rhizosphere microbiome to suppress soil-borne pathogens in field-grown tomato crops was also analysed. We discuss likely mechanisms underpinning microbiome-mediated tolerance to biotic stress in tomato crops and highlight research gaps to be considered in future investigations. Identifying functionally beneficial rhizosphere microbiota in healthy tomato crops may provide new insights into understanding plant–pathogen interactions and allow new strategies for exploring disease control.

Introduction: Through the combined use of two nitrification inhibitors, Dicyandiamide (DCD) and chlorate with nitrogen amendment, this study aimed to investigate the contribution of comammox Nitrospira clade B, ammonia oxidizing bacteria (AOB) and archaea (AOA) to nitrification in a high fertility grassland soil, in a 90-day incubation study.

Methods: The soil was treated with nitrogen (N) at three levels: 0 mg-N kg-1 soil, 50 mg-N kg-1 soil, and 700 mg-N kg-1 soil, with or without the two nitrification inhibitors. The abundance of comammox Nitrospira, AOA, AOB, and nitrite oxidising bacteria (NOB) was measured using qPCR. The comammox Nitrospira community structure was assessed using Illumina sequencing.

Results and Discussion: The results showed that the application of chlorate inhibited the oxidation of both NH4+ and NO2- in all three nitrogen treatments. The application of chlorate significantly reduced the abundance of comammox Nitrospira amoA and nxrB genes across the 90-day experimental period. Chlorate also had a significant effect on the beta diversity (Bray-Curtis dissimilarity) of the comammox Nitrospira clade B community. Whilst AOB grew in response to the N substrate additions and were inhibited by both inhibitors, AOA showed little or no response to either the N substrate or inhibitor treatments. In contrast, comammox Nitrospira clade B were inhibited by the high ammonium concentrations released from the urine substrates. These results demonstrate the differential and niche responses of the three ammonia oxidising communities to N substrate additions and nitrification inhibitor treatments. Further research is needed to investigate the specificity of the two inhibitors on the different ammonia oxidising communities.
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Leptosphaeria maculans and L. biglobosa are major pathogens of brassica crops, including canola (oilseed rape) and swede. Selection of canola cultivars with resistance genes to correspond with the avirulence genes of the local fungal population is an approach used overseas for controlling the disease. This study aimed to determine the main causal agent(s) in New Zealand, and, for a subset of 30 L. maculans isolates, the presence of 7 avirulence alleles (AvLm1, AvLm2, AvLm4, AvLm5, AvLm6, AvLm7, and AvLm11). The pathogenicity of these isolates on canola ‘Flash’ and swede ‘Highlander’ was also determined. Leptosphaeria maculans was the predominant species (n=127) recovered from leaf lesions, stem canker and dry rot tissue on canola, swede and turnip plants, with only 4 isolates identified as L. biglobosa recovered from stem cankers on canola, cauliflower and kale. All avirulence alleles were present in the New Zealand L. maculans population, with AvLm5 and AvLm6 amplified from all 30 isolates. The most common allele structure was avLm1-avLm2-avLm4-AvLm5-AvLm6- AvLm7-avLm11 (n=15, Avr refers to avirulence and avr refers to virulence allele) representing isolates recovered from canola, swede and turnip across New Zealand, followed by avLm1-avLm2-AvLm4- AvLm5-AvLm6-AvLm7-AvLm11 (n=7) only recovered from canola from Canterbury. L. maculans isolates were more pathogenic on swede ‘Highlander’ than canola ‘Flash’, forming more extensive stem cankers on swede after 65 days. There was no correlation between pathogenicity of the isolates and crop origin, or avirulence groupings observed. This study provides a base line evaluation of the avirulence allele frequencies in the New Zealand L. maculans population.

The objectives of this study were to investigate the abundance and community composition of comammox Nitrospira under: (i) pasture-based dairy farms from different regions, and (ii) different land uses from the same region and soil type. The results clearly showed that comammox Nitrospira were most abundant (3.0 × 106 copies) under the west coast dairy farm conditions, where they were also significantly more abundant than canonical ammonia oxidisers. This was also true in the Canterbury dairy farm. The six land uses investigated were pine monoculture, a long term no input ecological trial, sheep + beef and Dairy, both irrigated and non-irrigated. It was concluded that comammox Nitrospira was most abundant under the irrigated dairy farm (2.7 × 106 copies). Contrary to the current industry opinion, the relatively high abundance of comammox Nitrospira under fertile irrigated dairy land suggests that comammox Nitrospira found in terrestrial ecosystems may be copiotrophic. it was also determined that comammox Nitrospira was more abundant under irrigated land use than their non-irrigated counterparts, suggesting that soil moisture is a key environmental parameter influencing comammox abundance. Comammox abundance was also positively correlated with annual rainfall, further supporting this theory. Phylogenetic analysis of the comammox Nitrospira detected determined that 17 % of the comammox community belonged to a newly distinguished subclade, clade B.2. The remaining 83 % belonged to clade B.1. No sequences from clade A were found.

The purpose of developing this high throughput assay was to determine whether there was evidence of pH adaptation in strains of rhizobia which nodulate subterranean clover (SC) and white clover (WC), and whether this was related to the pH of the soil of origin. pH is a first-order factor influencing the niche preferences of soil microorganisms and has been convincingly shown to be a key driver of soil bacterial communities. Naturalised strains of Rhizobium spp. that are pH-adapted may have the potential to better compete and/or persist in acidic or alkaline soils compared with introduced commercial strains. Three pilot studies were conducted to design the optimised bioassay. This bioassay tested the effect of pH-amended yeast mannitol broth (seven pH values from pH 4.5–9.0), across three time points, on the in vitro growth of 299 Rhizobium strains isolated from the nodules of SC and WC. The media pH where strains demonstrated fastest growth was related to the pH of the soil that strains were isolated from. However, the correlation between media pH and soil pH was strongly influenced by the growth of strains from alkaline soils (alkaline adaptation), especially in strains isolated from SC nodules.
•Development of a cheap, effective and highly replicable bioassay.•Growth of 299 Rhizobium strains measured in 7 pH broths over 48 h.•Strains tended toward alkaline adaptation, especially those from sub. clover.•Potential to develop some strains into new clover inoculants.

Grapevine trunk diseases (GTDs) are a threat to grape production worldwide, with a diverse collection of fungal species implicated in disease onset. Due to the long-term and complex nature of GTDs, simultaneous detection of multiple microbial species can enhance understanding of disease development. We used DNA metabarcoding of ribosomal internal transcribed spacer 1 (ITS1) sequences, supported by specific PCR and microbial isolation, to establish the presence of trunk pathogens across 11 vineyards (11-26 years old) over three years in Marlborough, the largest wine producing region in New Zealand. Using a reference database of trunk pathogen sequences, species previously associated with GTD, such as Cadophora luteo-olivacea, Diplodia seriata, Diplodia mutila, Neofusicoccum australe, and Seimatosporium vitis, were identified as highly represented across the vineyard region. The well-known pathogens Phaeomoniella chlamydospora and Eutypa lata had especially high relative abundance across the dataset, with P. chlamydospora reads present between 22 and 84% (average 52%) across the vineyards. Screening of sequences against broader, publicly available databases revealed further fungal species within families and orders known to contain pathogens, many of which appeared to be endemic to New Zealand. The presence of several wood-rotting basidiomycetes (mostly Hymenochaetales) was detected for the first time in the Marlborough vineyard region, notably, the native Inonotus nothofagii which was present at 1-2% relative abundance in two vineyards.

Grapevine trunk diseases (GTDs) are a significant problem for New Zealand viticulture. Endophytic actinobacteria are of interest as potential biocontrol agents due to their ability to inhibit plant pathogens and improve plant growth. However, no studies have investigated the diversity of actinobacteria associated with grapevines in New Zealand vineyards and their bioactivity. Actinobacteria diversity in different ‘Sauvignon blanc’ vine tissues from three vineyards (conventional and organic management, and different vine ages) was assessed using different methods and media. Forty-six endophytic actinobacteria were isolated, with more isolates recovered from roots (n = 45) than leaves (n = 1) and shoot internodes (n = 0). More isolates were recovered from the organic (n = 21) than conventional (n = 8) vineyard, mature (25-year old; n = 21) than young (2-year old; n = 2) vines and using a tissue maceration technique (n = 40). Actinomycete Isolation Agar, International Streptomyces Project 2, and Starch Casein media were effective for actinobacteria isolation. Most of the isolates recovered belonged to Streptomyces, with one isolate identified as Mycolicibacterium. Forty isolates were assessed for antifungal activity and plant growth-promoting (PGP) characteristics. Of these, 13 isolates had antifungal activity against test GTD pathogens (Dactylonectria macrodidyma, Eutypa lata, Ilyonectria liriodendri, Neofusicoccum parvum, and N. luteum). Eighteen isolates exhibited more than one PGP trait; 25siderophore production (n = 25), phosphate solubilization (n = 6), and indole acetic acid production (n = 16). Two strains, Streptomyces sp. LUVPK-22 and Streptomyces sp. LUVPK-30, exhibited the best antifungal and PGP properties. This study revealed the diversity of culturable endophytic actinobacteria from grapevines in New Zealand vineyards and their biocontrol potential against GTD pathogens.

Grapevine trunk diseases are an economically important issue for the New Zealand viticulture industry. The limited control strategies effective against these pathogens and the increasing awareness of the potential environmental impacts of fungicide applications has led to an increased interest in identifying alternative control strategies. Endophytic actinobacteria are of particular interest due to their capacity to produce bioactive compounds that inhibit phytopathogens. However, there have been no studies in New Zealand to investigate the diversity and bioactivity of endophytic actinobacteria against grapevine trunk pathogens. This study aimed to determine the most effective method and media for isolation of endophytic actinobacteria from different grapevine tissues. Grapevine tissues (leaf, stem and roots) were sampled from Sauvignon blanc vines in both a conventionally and an organically managed vineyard at Lincoln University. Two isolation techniques were tested on surface sterilised tissue: 1) direct plating of tissue pieces (2 mm²); and 2) tissue maceration, with tissue (0.1 g) homogenized in 1 mL water and 1520 metal beads (2 mm diam.). The tissue samples were plated on four media, International Streptomyces Project 2 agar (ISP2), Starch Casein agar (SC), Tap Water Yeast Extract agar (TWYE) and Actinomycete Isolation agar (AIA). The recovered actinobacteria isolates were identified based on morphology and sequencing of the 16S rRNA gene.
More isolates were recovered from roots (22 isolates), than leaves (1 isolates) or stems (0 isolates), with more actinobacteria isolated from the conventionally managed vineyard and using the tissue maceration technique. AIA (8 isolates), ISP2 (7 isolates), SC (5 isolates), and TWYE (3 isolates) were effective at isolating actinobacteria from grapevine tissues. Isolates identified as Streptomyces spp. (22 isolates) and Mycolicibacterium sp. (1 isolate) were recovered. Future work will use these methods to determine endophytic actinobacteria diversity associated with grapevines, and their ability to inhibit key grapevine trunk pathogens.

Pythium species are recognised as important soilborne plant pathogens, however, some of these species (mycoparasitic Pythium species) are aggressive parasites of other fungi and oomycetes. These species have potential as biological control agents. However, there is limited information regarding the population diversity of mycoparasitic Pythium species present in New Zealand, especially associated with socio-economically important horticultural crops such as grapevines. The objective of this study was to determine the best method to isolate mycoparasitic Pythium species from soil collected from the Lincoln University vineyards. Three methods were tested: (1) soil dilution plating; (2) sclerotia baiting; and (3) pre-colonised fungal host baiting. (1) was done by plating 1:50 soil dilutions on Pythium selective media (CMA-PARP). (2) Sclerotinia sclerotiorum sclerotia were placed into soil water suspensions and the sclerotia transferred after 48 hr onto selective media. (3) 1.5 g of each soil sample was placed onto a pre-colonised host fungal culture plate of either Botrytis cinerea, Fusarium oxysporum, Ilyonectria liriodendra or Neofusicoccum parvum. The plates were observed microscopically after 7, 14 and 21 days for presence of characteristic oospores and isolation was carried out from any positive plates. Fusarium oxysporum pre-colonised plates and the sclerotia baiting method were the most effective at recovering mycoparasitic Pythium species from the soil samples. Isolates morphologically identified as Pythium oligandrum, as well as other unidentified mycoparasitic Pythium species were recovered. No mycoparasitic Pythium species were recovered on B. cinerea, I. liriodendra or N. parvum pre-colonised plates, with Trichoderma spp. and Clonostachys spp. recovered on B. cinerea and N. parvum plates. The species identity of the recovered isolates are currently being confirmed using Sanger DNA sequencing. These methods will be used to determine the diversity of mycoparasitic Pythium species in New Zealand vineyards.