Characterisation of Pseudomonas syringae isolated from systemic infection of zucchini in Australia

Zucchini plants, with symptoms including twisted petioles, necrotic leaves, crown-rot and internal fruit-rot, were found in Bundaberg, Australia at a commercial field for the first time during late autumn 2016, resulting in direct yield losses of 70 to 80%. Three Pseudomonas syringae strains that isolated from symptomatic leaf (KL004-k1), crown (77-4C) and fruit (KFR003-1) were characterised and their pathogenicity evaluated on pumpkin, rockmelon, squash and zucchini. Biochemical assays showed typical results for P. syringae. The three isolates differed, however, in that two produced fluorescent pigment (KFR003-1 and 77-4C) whilst the third, KL004-k1, was non-florescent. Multi-locus sequence analysis classified the isolates to phylogroup 2b. The SNP analysis of core genome from the Australian and closely related international isolates of P. syringae showed two separate clusters. The Australian isolates were clustered based on fluorescent phenotype. Pathogenicity tests demonstrated all three isolates moved systemically within the inoculated plants and induced necrotic leaf symptoms in zucchini plants. Their identities were confirmed with specific PCR assays for P. syringae and phylogroup 2. Pathogenicity experiments also showed the Eva variety of zucchini was more susceptible than Rosa for all three isolates. Isolate KL004-k1 was more virulent than 77-4C on pumpkin, rockmelon, squash and zucchini. This study expands the knowledge of P. syringae isolates that infect cucurbits and provides useful information for growers about the relative susceptibility of a range of cucurbit species.

First Report of Internal Fruit Rot of Sweet Pepper in Korea Caused by Fusarium lactis

In June 2010, an internal fruit rot of sweet pepper (Capsicum annuum L.) fruit was found in a commercial greenhouse in Ilsan City, Korea. Disease incidence reached approximately 5% of 30 tons of harvested peppers. Affected fruits commonly did not show external symptoms. However, when the fruit was cut open, an internal rot and pinkish gray mycelium were observed on the seeds and the inner surface of fruit. Discolored soft patches, browning, and necrosis were observed on the outer surface of some fruits. Fragments (5 × 5 mm2) of the affected tissues were surface sterilized with 1% NaOCl for 30 s and then rinsed twice in sterile distilled water. The pieces were placed on water agar and incubated at 25°C for 4 to 6 days. Twenty-nine Fusarium isolates were obtained from 12 diseased fruits and maintained on synthetic low nutrient agar (SNA) at 10°C. The isolates were cultured on carnation leaf agar (CLA) and SNA at 23°C with 12 h of near-ultraviolet light per day for 14 days. Microconidia were abundant, borne in short, zig-zag chains or false heads, obovoid or clavate with a flattened base, and measured 4.3 to 7.1 × 2.2 to 3.3 μm. Macroconidia were sparse, thin walled, slender, straight to slightly curved, and measured 32 to 48 × 2.8 to 3.9 μm. Sporodochia were rare on CLA and chlamydospores were absent. The translation elongation factor 1-alpha (EF-1α) gene was amplified from four isolates (SPF01, SPF09, SPF16, and SPF22) by PCR assay using ef1 and ef2 primers (2), and the 700-bp amplification products were sequenced. The nucleotide sequences were deposited in GenBank (Accession Nos. JF411956 to JF411959). BLAST analysis showed 98% homology with the EF-1α sequence of Fusarium lactis NRRL25200 (GenBank Accession No. AF160272). All isolates were identified as F. lactis based on morphological and molecular characteristics (1). Pathogenicity tests of the four isolates were conducted by inoculating flowers on plants of the orange pepper cv. Orange Glory (3). A spore suspension was prepared by flooding 5-day-old cultures on potato dextrose agar with sterile distilled water. When the plants started to flower, each flower was inoculated by placing 20 μl of spore suspension (1 × 106 conidia/ml) on each flower. Four isolates of F. lactis were each inoculated onto three flowers on each of seven plants. Flowers from the same number of plants inoculated with sterile distilled water were used as the control treatment. Inoculated plants were kept in a greenhouse at 25°C by day and 20°C by night. Sixty days after inoculation, mature fruits were harvested and cut open to check for internal rot. Approximately 70% of inoculated fruits showed internal rot and pinkish gray mycelial growth on the inner surface of the fruits. No symptoms were observed on the control fruits. Fungal cultures resembling F. lactis were reisolated from inoculated fruits for all four isolates, fulfilling Koch's postulates. F. lactis has been reported on sweet pepper in the Netherlands and Canada (3). To our knowledge, this is the first report of internal fruit rot of sweet pepper caused by F. lactis in Korea. Although disease severity was low in this greenhouse, the economic impact on sweet pepper could be significant because the disease can reduce the quality, quantity, and market value of pepper fruits. References: (1) H. I. Nirenberg and K. O'Donnell. Mycologia 90:434, 1998. (2) K. O'Donnell et al. Proc. Natl. Acad. Sci. USA 95:2044, 1998. (3) J. Yang et al. Can. J. Plant Pathol. 31:47, 2009.