Search

Xylella fastidiosa is a xylem‐limited bacterial plant pathogen transmitted by insect vectors. It infects a wide range of plant species and causes devastating diseases. Botanical gardens are global repositories of plant diversity exposed to local biotic and abiotic stresses. We used molecular diagnostic tools for the detection of X. fastidiosa in a collection of New Zealand indigenous plants grown in a X. fastidiosa‐infected Californian area to determine if any species were infected with this bacterial pathogen and to help inform biosecurity responses. To this end, 130 New Zealand indigenous plant species comprising 72 genera, growing in the University of California Botanical Garden at Berkeley, California, were screened for X. fastidiosa. Multiple PCR‐based methods were used to detect the pathogen at the subspecies and sequence‐type levels directly from plant material; nine plant species tested positive by at least two PCR‐based methods. All nine infections were identified as X. fastidiosa subsp. multiplex sequence types 6 or 7 by the amplification of seven housekeeping genes. Three strains were cultured in vitro and their whole genome sequences were obtained; these strains belonged to three distinct clades within subspecies multiplex, indicating that the infections were not transmitted among these New Zealand indigenous plant species. The information gathered in this study will help to assess the risk of X. fastidiosa to New Zealand indigenous flora and the potential spillover to crops of agricultural importance. The work also shows the applied value of botanical garden collections as sentinels for international plant pathogen biosecurity risk assessment. [ABSTRACT FROM AUTHOR]

Previously genetically characterised strains of Pseudomonas syringae. pv. syringae (Pss), [P. amygdali pv. morsprunorum] (Pam, syn. P. s. pv. morsprunorum race 1) and Pseudomonas spp. from New Zealand were characterised for their pathogenicity and aggressiveness in plant tissue and associated virulence factors. Lesions on detached, Pss‐inoculated immature fruit increased rapidly in size and, at 10 days post inoculation (dpi), had larger areas under the disease progress curve (AUDPC) than Pam‐inoculated fruit (48.9 and 22.0, respectively). Detached leaves infiltrated with Pss‐developed symptoms within 1 dpi and from 2 dpi for Pam. Necrosis from most Pss strains extended into the leaf veins by 7 dpi, while Pam strains' necrosis was confined to the inoculation site. On detached 1‐year‐old cherry shoots, Pseudomonas spp. strains exhibited the smallest mean lesion size (2.1–2.4 mm), whereas larger mean lesion sizes were observed with Pss strains (5.7–13.7 mm) and Pam strains (3.9–14.0 mm). A functional T3SS was inferred for Pss and Pam strains based on the hypersensitivity reactions observed on tobacco leaves and symptoms elicited on cherry tissue. Syringomycin production was prevalent (88%) among Pss strains. In contrast, only 1.4% of Pam strains produced coronatine. Most Pss strains (97.0%) were able to catalyse ice formation. The coexistence of strains with varying degrees of virulence and non‐pathogenic strains suggests a complex ecological balance, where multiple factors, including genetic variation, virulence traits and environmental conditions, shape the population dynamics and disease outcomes. [ABSTRACT FROM AUTHOR]

Bacterial canker of cherry, caused by Pseudomonas syringae pathovars, is a major constraint to cherry growing in New Zealand. The prevalence of strains from cherry orchards in Central Otago, the main growing area for cherries in New Zealand, was studied, to better understand the epidemiology of the disease. Pseudomonas spp. isolates were collected from symptomatic and asymptomatic cherry tissue from 23 commercial cherry orchards in 2015. Isolates were classified into strains belonging to three different taxonomic groups by determining their phylogeny using the gltA gene sequence for all the strains and multilocus sequence analysis (MLSA) of four housekeeping genes for 35 strains. Pathogenicity of all Central Otago strains was tested on immature cherry fruit to support the phylogenetic classification. The two main taxonomic groups were P. syringae pv. syringae (Pss) and P. syringae pv. morsprunorum race 1 (Psm1), in Phylogroup 2 (PG2) and Phylogroup 3 (PG3), respectively. The third group comprised nonpathogenic strains classified as Pseudomonas spp. Strains of Psm1 formed a monophyletic group, representing an almost clonal population. There was more variation detected within strains of Pss, although they were restricted to group PG2d. Nonpathogenic Pseudomonas spp. and pathogenic Pss and Psm1 strains coexisted in the same orchard. It was concluded that Pss is the predominant pathovar in Central Otago. This is the first detailed study of the P. syringae species complex in cherry orchards in New Zealand and provides the basis for future epidemiology studies. [ABSTRACT FROM AUTHOR]

Bacterial pathogens are major causes of crop diseases, leading to significant production losses. For instance, kiwifruit canker, caused by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), has posed a global challenge to kiwifruit production. Treatment with copper and antibiotics, whilst initially effective, is leading to the rise of bacterial resistance, requiring new biocontrol approaches. Previously, we isolated a group of closely related Psa phages with biocontrol potential, which represent environmentally sustainable antimicrobials. However, their deployment as antimicrobials requires further insight into their properties and infection strategy. Here, we provide an in‐depth examination of the genome of ΦPsa374‐like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo‐electron microscopy of ΦPsa374, we revealed the structural proteome and that this phage possess a T = 9 capsid triangulation, unusual for myoviruses. Furthermore, we show that ΦPsa374 phage resistance arises in planta through mutations in a glycosyltransferase involved in LPS synthesis. Lastly, through in vitro evolution experiments we showed that phage resistance is overcome by mutations in a tail fibre and structural protein of unknown function in ΦPsa374. This study provides new insight into the properties of ΦPsa374‐like phages that informs their use as antimicrobials against Psa. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafoodprocessing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants. IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes. [ABSTRACT FROM AUTHOR]

Molecular detection of phytopathogens is increasingly being applied to identify regulated organisms at the border in many parts of the world. However, even with molecular tests, complete phenotyping and identification of a strain is often time consuming and sometimes inconclusive. In this study, a leaf‐based pathogenicity test was used to separate pseudomonads into two groups, Group A containing pathogens, and Group B containing saprotrophs. Comparative genomics of 56 pseudomonad genomes from different plant hosts (including 29 strains from kiwifruit) agreed with kiwifruit pathogenicity test results, placing pathogens into Group A and saprotrophs into Group B. Sixteen loci were found unique to Group A. A PCR assay was developed for amplification of one of these loci, the trehalose phosphatase gene. The generation of this 655 bp amplicon was associated with production of water‐soaked lesions on inoculated kiwifruit leaves by pseudomonads in Group A. This test was validated for further strains from all seven pathogenic Pseudomonas phylogroups, non‐pathogenic pseudomonads, and other bacterial genera. The sensitivity of the PCR was comparable to the limit of recovery of pseudomonads by culturing. This simple PCR assay could be used as part of a testing pipeline at the border and for general surveillance for screening plants with and without symptoms, offering the potential to detect uncharacterized pseudomonads that may pose a biosecurity risk. The method was shown to be able to rapidly identify pathogens cultured from plant material with symptoms, or, more importantly, to detect pathogens directly from plant tissue. [ABSTRACT FROM AUTHOR]

The ITS1-5.8S-ITS2 rRNA (internal transcribed spacer (ITS)) region is used for examining the phylogenetic relationships of Rhizopogon roseolus and other closely related fungi belonging to Rhizopogon subgenus Roseoli in order to determine the genetic basis for phenotypic differences among R. roseolus isolates from different geographic regions. The ITS region is used for designing a multiplex PCR for the simultaneous identification of Japanese and New Zealand R. roseolus isolates in order to track the establishment of ectomycorrhiza on Pinaceae radiate seedlings inoculated with commercially valuable R. roseolus.

We present an account of Rhizopogon introduced from plantings of exotic pine plantations in Argentine Patagonia. Nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) and nuc 28S rDNA (28S) sequences were used to identify specimens from Argentina and examine their relationships with geographically different Rhizopogon species. Based on phylogenetic analyses, we confirm that four species of Rhizopogon occur in pine plantations across Patagonia. Several Rhizopogon collections from Pinus ponderosa plantations across different provinces cluster with R. arctostaphyli, a species within R. subg. Amylopogon. The majority of Patagonian Rhizopogon, however, form three different lineages in R. subg. Roseoli. The first of these, R. roseolus sensu Trappe, includes numerous collections from Pinus ponderosa, P. contorta, and P. radiata stands of North American affiliation. The second, R. roseolus sensu Martin and Garcia from P. ponderosa plantations, clusters in clade IIIa of the R. roseolus complex, which also includes the holotype collection of R. mohelnensis from the Czech Republic. The third species in R. subg. Roseoli, and fourth species overall from Patagonia, is R. granuloflavus from Pinus ponderosa plantations. Multiplex polymerase chain reaction (PCR) of numerous Roseoli samples failed to produce an amplicon indicative of either Japanese or New Zealand shoro. [ABSTRACT FROM AUTHOR]

Morchella species are edible fungi in high demand and therefore command high prices in world markets. Phenotypic-based identification at the species-level remains inadequate because of their complex life cycles, minor differences and plasticity of morphological characteristics between species, and the lack of agreement between scientific and common names. In Patagonia–Argentina, morels are associated with native forests of Austrocedrus chilensis (Cordilleran or Chilean cypress) and Nothofagus antarctica (ñire) and several exotic conifers that were introduced from western North America. Little is known about their taxonomy and phylogenetic relationships with other species in the genus. This work focused on the identification of collections of Morchella from Patagonia and their phylogenetic relationships with other species from the Northern Hemisphere. The comparison was made by analysis of DNA sequences obtained from four loci: the nuclear ribosomal internal transcribed spacer region (ITS) and the partial RNA polymerase I gene ( RPB1 ) for the complete collection; and ITS, RPB1 , RNA polymerase II gene ( RPB2 ), and translation elongation factor ( EF1-α ) for the species-rich Elata Subclade. Analyses of individual and combined data sets revealed that Patagonian morels belong to the Elata Clade and comprised three strongly supported species-level lineages from both Patagonian native forest, and exotic trees introduced from western North America. One lineage was identified as Morchella frustrata phylogenetic species Mel-2 , which is known from the USA and Canada. The second lineage, which appeared to be ‘fire-adapted’, was identified as Morchella septimelata phylogenetic species ( Mel-7 ), which is also known from the USA. This species was collected from burned native forests mainly composed of A. chilensis and N. antarctica but also Pseudotsuga menziesii ( Mirb.) Blanco, which is native to western North America. The phylogenetic analyses suggested that the third species from Patagonia was nested within the species-rich Elata Subclade and represents a new species-level lineage (informally designated Mel-37 ) within Elata Clade. The present collections from Patagonia constitute the southernmost latitude from which Morchella has been reported to date. The identification of two Argentine morels as North American taxa is therefore a remarkable biogeographic pattern. In view of the hypothesis that the Elata Clade originated in western North America, we speculate that at least two of the lineages colonized South America from North America via long distance dispersal, migration or, more likely, they were introduced with the exotic tree species that they were collected near. [ABSTRACT FROM AUTHOR]

Pseudomonas syringae pv. actinidiae is a reemerging pathogen which causes bacterial canker of kiwifruit (Actinidia sp.). Since 2008, a global outbreak of P. syringae pv. actinidiae has occurred, and in 2010 this pathogen was detected in New Zealand. The economic impact and the development of resistance in P. syringae pv. actinidiae and other pathovars against antibiotics and copper sprays have led to a search for alternative management strategies. We isolated 275 phages, 258 of which were active against P. syringae pv. actinidiae. Extensive host range testing on P. syringae pv. actinidiae, other pseudomonads, and bacteria isolated from kiwifruit orchards showed that most phages have a narrow host range. Twenty-four were analyzed by electron microscopy, pulse-field gel electrophoresis, and restriction digestion. Their suitability for biocontrol was tested by assessing stability and the absence of lysogeny and transduction. A detailed host range was performed, phage-resistant bacteria were isolated, and resistance to other phages was examined. The phages belonged to the Caudovirales and were analyzed based on morphology and genome size, which showed them to be representatives of Myoviridae, Podoviridae, and Siphoviridae. Twenty-one Myoviridae members have similar morphologies and genome sizes yet differ in restriction patterns, host range, and resistance, indicating a closely related group. Nine of these Myoviridae members were sequenced, and each was unique. The most closely related sequenced phages were a group infecting Pseudomonas aeruginosa and characterized by phages JG004 and PAK_P1. In summary, this study reports the isolation and characterization of P. syringae pv. actinidiae phages and provides a framework for the intelligent formulation of phage biocontrol agents against kiwifruit bacterial canker. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a foodborne human pathogen that causes systemic infection, fetal-placental infection in pregnant women causing abortion and stillbirth and meningoencephalitis in elderly and immunocompromised individuals. This study aimed to analyse L. monocytogenes from different sources from New Zealand (NZ) and to compare them with international strains. We used pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole-genome single nucleotide polymorphisms (SNP) to study the population structure of the NZ L. monocytogenes isolates and their relationship with the international strains. The NZ isolates formed unique clusters in PFGE, MLST and whole-genome SNP comparisons compared to the international isolates for which data were available. PFGE identified 31 Asc I and 29 Apa I PFGE patterns with indistinguishable pulsotypes being present in seafood, horticultural products and environmental samples. Apart from the Asc0002:Apa0002 pulsotype which was distributed across different sources, other pulsotypes were site or factory associated. Whole-genome analysis of 200 randomly selected L. monocytogenes isolates revealed that lineage II dominated the NZ L. monocytogenes populations. MLST comparison of international and NZ isolates with lineage II accounted for 89% (177 of 200) of the total L. monocytogenes population, while the international representation was 45.3% (1674 of 3473). Rarefaction analysis showed that sequence type richness was greater in NZ isolates compared to international trend, however, it should be noted that NZ isolates predominantly came from seafood, horticulture and their respective processing environments or factories, unlike international isolates where there was a good mixture of clinical, food and environmental isolates. • The set of New Zealand Listeria monocytogenes food isolates formed unique genetic clusters. • There were 23 clonal complexes and 25 sequence types found in this study. • NZ horticulture and seafood L. monocytogenes isolates were predominantly lineage II accounting for 89%. • WGS-SNP gave unambiguous and more robust phylogenetic inferences than PFGE or MLST [ABSTRACT FROM AUTHOR]

The draft genome sequences of three " Candidatus Symbiopectobacterium" isolates that were collected from New Zealand-grown potato tubers represent the first report of this proposed taxon in the Southern Hemisphere. Their symbiosis with insects and nematodes and their presence on plants may lead to new strategies for pest control and crop management.

We present here the draft genome sequences of 14 Pseudomonas strains isolated from Prunus sp. plants in New Zealand and overseas. These new genomic data will be used to improve the detection of Pseudomonas strains found in imported plant material at the New Zealand border, improving the time involved in the process of biosecurity decision-making., (© Crown copyright 2018.)

Pectobacterium carotovorum subsp. actinidiae is the causal agent of summer canker in kiwifruit plants in South Korea. We report here the draft genome sequences of two P. carotovorum subsp. actinidiae strains, ICMP 19971 and ICMP 19972, which were originally isolated from Actinidia chinensis with symptoms of summer canker. These genome sequences will aid in the identification of genetic traits associated with their unusual capacity to cause canker and help understanding of the threat these exotic enterobacteria pose to the New Zealand kiwifruit industry., (Copyright © 2017 Visnovsky et al.)

In this paper, we present the draft sequences of 18 genetically diversePseudomonasstrains isolated from kiwifruit plants in New Zealand and overseas, including a number that are currently not fully characterized. These sequences will aid in the diagnosis ofPseudomonason kiwifruit for future pest management and border security decision-making., (Copyright © 2016 Visnovsky et al.)