A Rapid qPCR for the Detection of Verticillium nonalfalfae MLST2 - A Highly Pathogenic Fungus on Kiwifruit.

A highly pathogenic fungus characterized as Verticillium nonalfalfae multilocus sequence type 2 (MLST2) is an emerging fungal pathogen causing Verticillium wilt on kiwifruit. Although V. nonalfalfae MLST2 has not been reported outside Chile, there is a risk that this pathogen could spread through the global movement of germplasms to other countries. Current diagnostic methods for this fungus rely on a laborious and time-consuming plating assay for morphological identification and DNA sequence analysis. In this study, we describe the development and validation of a novel quantitative polymerase chain reaction (qPCR) assay for rapid and specific detection of V. nonalfalfae MLST2 in plant tissues. The assay targets the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and was shown to detect all tested isolates of V. nonalfalfae MLST2 with a detection limit of approximately 2 pg of pathogen genomic DNA. There was no cross-reaction with V. nonalfalfae MLST1, other Verticillium species, or non-target fungal species found on kiwifruit. This assay was duplexed with a plant internal control for simultaneous detection of the pathogen and cytochrome oxidase gene from the host plant. This new specific and sensitive qPCR assay is a valuable molecular diagnostic tool for rapid screening of imported plant material and would also be useful for testing samples collected from field surveillance activities to monitor the presence of V. nonalfalfae MLST2.

A New TaqMan Real-Time PCR Assay for Detecting the Blueberry Pathogen Monilinia vaccinii-corymbosi.

Monilinia vaccinii-corymbosi (Mvc) is an important fungal pathogen of blueberry, causing mummy berry disease. While the symptoms of the advanced stages of the disease can be obvious, diagnosing its early stages can be challenging. To facilitate fast and sensitive screening of asymptomatic or latently infected plant material for Mvc, we developed a specific TaqMan real-time PCR assay targeting the internal transcribed spacer (ITS) region. The assay was shown to be specific to Mvc and did not cross react with any of the other tested Monilinia species or other blueberry pathogens. Using the multicopy ITS region ensured high analytical sensitivity, enabling very low concentrations of Mvc DNA (0.1 pg) to be detected both in water and host DNA matrix. Comparable results were obtained in interlaboratory testing, showing that the assay is robust, and can be effectively used in other laboratories. Assay sensitivity was also confirmed on infected plant tissue, showing that it is effective in detecting the pathogen in infected asymptomatic stem tissue, as well as infected tissue that was mixed with healthy tissue at a ratio of 1:10 by weight. The assay was duplexed with a plant internal control (cytochrome oxidase gene) for simultaneous amplification of the pathogen and plant internal control in a single reaction. This new diagnostic tool can be used for sensitive and rapid screening of blueberry plants for the presence of Mvc in many different settings, e.g., for breeding programs, research, or biosecurity diagnostics.

A New Real-Time PCR Assay for Detecting Fungi in Genus Ceratocystis.

The genus Ceratocystis contains several significant plant pathogens, causing wilt and canker disease on a wide range of plant species. There are >40 known species of Ceratocystis, some of which are becoming increasingly important in agricultural or natural ecosystems. The diagnostic procedures for most Ceratocystis species rely on time-consuming and labor-intensive culturing approaches. To provide more time-efficient and sensitive molecular diagnostic tools for Ceratocystis, a generic TaqMan real-time PCR assay was developed using the ITS gene. This novel two-probe TaqMan assay amplified DNA from all tested Ceratocystis species. Some nonspecific amplification of a few species from closely related genera was observed under certain conditions; however, these false-positive detections could be ruled out using the additional PCR primers developed for further sequence-based identification of the detected species. The assay was found to be highly sensitive, as it detected 0.2 pg/µl of Ceratocystis DNA in water as well as in host DNA matrix. Further validation with artificially inoculated fig stem tissue demonstrated that the assay was also able to effectively detect the pathogen in infected asymptomatic stem tissue. This newly developed real-time PCR assay has practical applications in biosecurity, conservation, and agriculture; it will enable the detection of Ceratocystis species directly from plant material to facilitate more sensitive screening of imported plant germplasm, and allow rapid tracking of pathogens in the case of disease outbreaks.

First report of Colletotrichum fructicola, C. perseae, and C. siamense causing anthracnose disease of avocado (Persea americana) in New Zealand.

In January and March 2019, an inspection of 11 commercial 'Hass' avocado orchards in mid-North and Tauranga (New Zealand) was conducted by NZ Avocado Growers Association Inc. (NZAGA) and the samples were sent to Plant Diagnostics Limited for investigation of a newly observed fruit staining symptom termed "tannin stain". Fruit symptoms consisted of areas of minute small spots which coalesced into areas of tear staining associated with water movement over the fruit's surface (Supplementary Fig. 1). Up to seven trees per orchard were sampled targeting symptomatic fruit with the aim of determining the cause of the problem. Fruit was surface disinfected for 4 minutes in 1% sodium hypochlorite solution and sections from lesions were plated on agar medium (prune extract agar) to isolate any plant pathogens. The predominant fungi isolated, represented species in the Colletotrichum acutatum, C. gloeosporioides, and C. boninense species complexes. Since the morphological characters within these complexes overlap (see Supplementary Fig. 2 for examples), the isolates were differentiated by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene and, where necessary, the calmodulin (CAL) gene and/or the Apn2-Mat1-2 intergenic spacer region (ApMat) locus (Weir et al., 2012; Rojas et al., 2010). The sequence analysis revealed eight Colletotrichum species comprising C. alienum, C. aotearoa, C. cigarro, C. fioriniae, C. fructicola, C. karstii, C. perseae, and C. siamense. This range included three species that have not previously been recorded in New Zealand: C. fructicola (Cf), C. perseae (Cp), and C. siamense (Cs). Colonies for all these three fungi were white to grey with salmon-coloured and black acervuli. Conidia were aseptate, hyaline, straight, cylindrical, with broadly rounded ends, forming on cylindrical conidiogenous cells. The respective GPDH, CAL, and/or ApMat sequences of the Cf, Cp, and Cs isolates were identical to reference sequences of representative isolates in GenBank (e.g. ApMat: Cf - KX620181, Cp - KX620177, Cs - KP703788). An isolate for each species is stored in the International Collection of Microorganisms from Plants (Cf - ICMP22409, Cp - ICMP22431, Cs - ICMP22411) and sequences are deposited in GenBank (accession numbers MT522858-MT522865). Pathogenicity of each of the newly recorded species was confirmed on freshly picked 'Hass' avocado fruit. After surface disinfection with 1% sodium hypochlorite solution for 5 minutes, fruit was triple washed with sterile water and air dried. Five fruits per species were pin-pricked and inoculated with 10µL of conidial suspension (7 x 106 to 1 x 107 conidia/mL) prepared with sterile water containing Tween 20 (1µL/mL H2O) from 6-day-old cultures grown on PDA. Control fruit was pin-pricked and mock-inoculated with sterile water containing Tween 20 (1µL/mL H2O). All fruit was incubated in moist chambers at 25°C for 7 days. The three Colletotrichum species produced anthracnose symptoms on inoculated fruit whereas no symptoms were observed on control fruit (Supplementary Fig. 3). Each one of the species was successfully re-isolated from symptomatic tissue and identified using the methods described above, fulfilling Koch's postulates. While Cf and Cs have been reported from several hosts and countries to date (Weir et al. 2012), Cp has only been found from avocado in Israel (Sharma et al. 2017) and grape in Japan (Yokosawa et al. 2020). Although a number of species from the C. gloeosporioides species complex, i.e. C. alienum, C. aotearoa, C. cigarro, and C. gloeosporioides have been previously associated with avocado diseases in New Zealand, the detections of Cf, Cp, and Cs represent first records. In this study, eight Colletotrichum species were associated with the "tannin stain" fruit symptoms in New Zealand avocado orchards. The individual contribution of the newly recorded pathogens Cf, Cp, and Cs to the observed disease symptoms was not determined, but their detection highlights the importance of sequence-based identification of Colletotrichum species, as morphology is insufficiently robust to separate cryptic species. Accurate identification of pathogens provides knowledge of species biodiversity that may be useful in biosecurity decision making. Since it has been reported that fungicide treatment efficiencies differ for some closely related Colletotrichum species on grape (Yokosawa et al. 2020), accurate identification might also contribute to establishing effective management strategies.

Draft Genome Sequence of Pseudomonas sp. Strain ICMP 22404, Isolated from Solanum lycopersicum Plants with Pith Necrosis Symptoms.

We report here the draft genome sequence of Pseudomonas sp. strain ICMP 22404, isolated from Solanum lycopersicum plants showing pith necrosis symptoms. The draft genome size is 6,686,400 bp, consisting of 86 contigs with a G+C content of 60.7% and containing 5,876 coding sequences, 60 tRNAs, and 11 rRNAs.

Isolation of Litylenchus coprosma from Coprosma macrocarpa, a new host and distribution in New Zealand.

Coprosma macrocarpa, known as the large-seeded coprosma or coastal karamu, is a shrub endemic to New Zealand. To our knowledge, no reports of plant parasitic nematodes associated with C. macrocarpa have been reported. Here we report the detection and identification of the nematode, Litylenchus coprosma, extracted from C. macrocarpa in Otata Island.

First record of the root knot nematode, Meloidogyne minor in New Zealand with description, sequencing information and key to known species of Meloidogyne in New Zealand.

Meloidogyne minor Karssen et al. 2004 was collected from perennial ryegrass (Lolium perenne L.) growing in a sports ground in Christchurch, New Zealand. This is a new record for M. minor, the first report of this nematode occurring in New Zealand, and the second report from the southern hemisphere (after Chile). In general, the New Zealand isolate of M. minor corresponds well to the descriptions of M. minor given by Karssen et al. (2004). The New Zealand isolate is characterized by having a female with dorsally curved stylet, 13-14 µm long, with transversely ovoid knobs slightly sloping backwards from shaft; rounded perineal pattern; and male with stylet 16-19 µm long, large transversely ovoid knobs sloping slightly backwards from shaft; head region not set off, labial disc elevated, lateral lips prominent; and second stage juvenile 370-390 µm long, with hemizonid posterior but adjacent to excretory pore; tail 53-63 µm long; and a distinct hyaline tail terminus 14-18 µm long. In addition, molecular phylogeny using near full length small subunit (SSU), D2/D3 expansion segments of the large subunit (LSU), the internal transcribed spacer region (ITS1 and 2), and the intergenic spacer (IGS2) of the ribosomal rDNA supports the identification.