Draft Genome Sequence of the Oomycete Globisporangium splendens Strain rgcb-1

Globisporangium splendens (formerly Pythium splendens) is an oomycete pathogen of many economically important vegetable crops. Here, we present the first draft genome of P. splendens, which comprises 197 scaffolds with a total length of 53.3 Mb and 17,350 predicted protein-coding genes.

Viability of Oomycete Propagules Following Ingestion and Excretion by Fungus Gnats, Shore Flies, and Snails

Sporangia of Phytophthora capsici and P. nicotianae, as well as hyphal swellings of Pythium splendens, P. sylvaticum, and P. ultimum, were ingested by adult shore flies but none were viable after passing through the digestive tract. Oospores of Pythium aphanidermatum retained their viability following ingestion by adult shore flies. Larval stages of fungus gnats and shore flies ingested sporangia of Phytophthora capsici, P. nicotianae, and P. ramorum, but they were not viable upon excretion. In contrast, hyphal swellings of Pythium splendens, P. sylvaticum, and P. ultimum, chlamydospores of Phytophthora ramorum, and oospores of Pythium aphanidermatum, retained their viability after passage through the digestive tract of these larvae. Snails were capable of ingesting and excreting viable sporangia and chlamydospores of P. ramorum, which upon excretion infected detached leaves. Although the impact of larvae and snails in the rapid dissemination of pathogen propagules is unknown, this work does highlight the possibility that some often-ignored animal–fungus interactions should be considered in long-range dispersal of pathogen propagules via food webs.

First Report of Pythium splendens Associated with Severe Wilt of Muskmelon (Cucumis melo) in Oman

Muskmelon (Cucumis melo L.) is one of the most important vegetable crops in Oman. In the fall of 2004, sudden wilt was observed in muskmelon grown in a field at Sultan Qaboos University, Muscat. The disease was characterized by rapid collapse of vines and muskmelon plants at the fruit production to maturation stage, associated with brown-to-dark brown rotted primary and secondary roots. The disease resulted in death of more than 85% of muskmelon plants in that field. On potato dextrose agar (PDA), with published methods (1), Pythium spp. were consistently isolated from crowns and roots of plants showing wilt symptoms. Further identification of five isolates of Pythium with sequences of the internal transcribed spacer (ITS) of the ribosomal DNA (1) using ITS1 and ITS4 primers produced a nucleotide sequence 806 bp long, which was identical among all isolates. Comparison with sequences deposited at the National Center for Biotechnology Information revealed 100% nucleotide similarity to a previously published sequence (Accession No. DQ381808) of isolate P091 of P. splendens from cucumber from Oman, for which identification has also been confirmed by morphological characteristics. The sequence of one isolate of P. splendens (P222) was assigned GenBank Accession No. EF546436 and deposited at CBS under Accession No. CBS121855. In pathogenicity tests conducted in a greenhouse, P. splendens induced damping-off symptoms on 7-day-old muskmelon seedlings and also reproduced the same wilt symptoms observed in the field when 2-month-old muskmelon plants were inoculated with 3-day-old P. splendens grown in PDA. To our knowledge, this is the first report of association of P. splendens with wilt of muskmelon in Oman. Reference: (1) A. M. Al-Sa'di et al. Plant Pathol. 56:140, 2007.

First Report of Formosan Michelia Seedling Root Rot Caused by Pythium splendens in Taiwan

Formosan michelia (Michelia compressa (Maxim.) Sargent) is a valuable evergreen tree in Taiwan that is distributed from low to medium (200 to 1,800 m) altitudes. In many nurseries in Taiwan, Formosan michelia seedlings grow poorly or wilt. The etiology of the disease observed in April 2004 in a nursery in Jinshan was investigated. Diseased seedlings with chlorotic leaves and decayed feeder roots lost leaves, died back, and then wilted. The putative pathogen, Pythium splendens Braun, was isolated and identified on a morphological basis (1). P. splendens was isolated from the roots of diseased seedlings on 2% water agar with 100 ppm of ampicillin. Isolates increased daily on potato dextrose agar at 24°C by 27 to 30 mm and on malt extract agar (MEA) by 23 to 25 mm. No zoosporangia and zoospores were produced. The main hyphae were as much as 9 μm wide on MEA. Hyphal swellings were abundant, globose, smooth, terminal, and 33 to 42 μm in diameter, often with dark, densely granulated contents. Attempted matings of four P. splendens isolates in V8 medium failed. To prove pathogenicity, the four isolates were cultured in 300-ml flasks containing 150 ml of 2% malt extract medium at room temperature for 14 days. The mycelia were homogenized in sterile water at 4,500 rpm for 5 min. The suspension was adjusted to 5 × 106 hyphal swellings per ml. Roots of the 2-month-old seedlings were immersed in the suspension for 2 h, whereas the control seedlings were immersed in sterilized water. Five seedlings of each of three replicates were inoculated with one of the four isolates for a total of 60 seedlings. Controls were replicated in the same way. The inoculated plants were transplanted into plastic flowerpots containing sterilized peat and moss and kept in the greenhouse at 20 to 24°C. After 14 days, inoculated seedlings developed symptoms like those of the original plants. The putative pathogen was reisolated from the roots of inoculated plants. Cultures are maintained at the Forest Pathology Lab of the National Taiwan University. To our knowledge, this is the first report of proof of pathogenicity of P. splendens on Formosan michelia seedlings. Reference: (1) A. J. Van der Plaats-Niterink. Stud. Mycol. 21:151, 1981.

First Report of Stem Rot of Guiana Chestnut (Pachira aquatica) Caused by Pythium splendens

Guiana chestnut is a perennial tropical plant that has recently become popular as a potted ornamental in Japan. In October 2001, severe stem rot occurred on Guiana chestnut plants grown in a greenhouse in Mie Prefecture, Japan. Water-soaked lesions appeared initially at the base of the stems and enlarged gradually toward the tops of plants. The affected tissues were softened and turned dark brown. Rotting was observed in the vascular bundles with advanced disease development. Globose hyphal swellings were numerous on diseased stems. Sections from diseased stems were cleaned by washing with running tap-water, placed on water agar, and incubated at 25°C. A species of Pythium was identified on the basis of morphological and cultural characteristics (1) and isolated consistently from the rotted stems of diseased plants. All isolates produced abundant hyphal swellings that were globose, smooth, 12 to 39 μm in diameter, mostly terminal, dark colored, and with dense granulated contents. Zoospores were absent. All isolates were of the compatibility ‘+ type’ with production of sexual organs when paired with cultures of the ‘- type’ tester isolate of Pythium splendens Braun (CBS462.48). Oogonia produced by crossings between Guiana chestnut isolates and isolate CBS462.48 were terminal or intercalary, globose, smooth-walled, and 32 to 38 μm in diameter. Antheridia were terminal, one to three per oogonium, sac-like, and diclinous. Oospores were single, aplerotic, globose, and 28 to 32 μm in diameter. The thickness of the oospore wall ranged from 1 to 2 μm. The internal transcribed spacer rDNA sequences of representative isolate OPU591 from Guiana chestnut matched those of CBS462.48 (similarity 99.2%) and have been deposited in GenBank under the Accession No. AY375242. Pathogenicity tests were conducted on potted Guiana chestnut plants (30 cm high and 7 to 10 cm in diameter at base of the stem) using isolate OPU591. A mycelial suspension from one culture, grown at 25°C for 7 days on water agar, was inoculated onto a single plant. Prior to inoculation, a wound (10 mm deep and 30 mm long) was made on the surface at the stem base on five plants. The mycelial suspension was poured onto the base of the stems of five wounded and five nonwounded plants. In addition, five wounded and five nonwounded, noninoculated plants were used as controls. Plants were maintained in a greenhouse for 8 weeks after inoculation. The temperature and relative humidity in the greenhouse ranged from 25 to 30°C and 65 to 75%, respectively. Dark-brown rotting developed on the stems of all wounded, inoculated plants by 20 days after inoculation. P. splendens was isolated from diseased tissues and found to be morphologically identical to the original isolate. This confirmed P. splendens as the causal agent of the disease. Disease did not develop on nonwounded inoculated plants or noninoculated plants. To our knowledge, this is the first report of P. splendens on Guiana chestnut. Potted plants of Guiana chestnut are often injured by frequent transplanting and transferring. Such injuries may have predisposed the plant to infection by P. splendens. Reference: (1) A. J. van der Plaats-Niterink. Page 1 in: Monograph of the Genus Pythium. Studies in Mycology Vol. 21, Centraalbureau Voor Schimmelcultures, Baarn, the Netherlands, 1981.

Involvement of Phenazines and Anthranilate in the Antagonism of Pseudomonas aeruginosa PNA1 and Tn5 Derivatives Toward Fusarium spp. and Pythium spp.

Pseudomonas aeruginosa PNA1, isolated from the rhizosphere of chickpea in India, suppressed Fusarium wilt of chickpea, caused by Fusarium oxysporum f. sp. ciceris, and Pythium damping-off of bean, caused by Pythium splendens. When grown in culture, PNA1 produced the phenazine antibiotics phenazine-1-carboxylic acid and oxychloraphine, and inhibited mycelial growth of F. oxysporum f. sp. ciceris, P. splendens, and certain other phytopathogenic fungi. Two mutants (FM29 and FM13) deficient in phenazine production were obtained following transposon mutagenesis of PNA1. The transposon in the genome of FM29 was localized to phnA, which is thought to encode a subunit of anthranilate synthase II involved in the phenazine biosynthesis. The FM13 mutation was complemented by trpC, which encodes indole glycerol phosphate synthase in the tryptophan biosynthesis pathway; consequently, FM13 could not grow on a minimal medium in the absence of tryptophan. Neither FM29 nor FM13 suppressed Fusarium wilt of chickpea to the level achieved by the wild-type strain, indicating that phenazine production contributed to the biocontrol of this disease by P. aeruginosa PNA1. FM29 was also less effective than the phenazine-producing parental strain in biological control of Pythium damping-off of bean, but FM13 was as effective as the parental strain in suppressing this disease. Anthranilate, an intermediate in the tryptophan biosynthesis pathway, suppressed mycelial growth of Pythium spp. in culture and Pythium damping-off of bean and lettuce. Anthranilate, excreted by FM13 as a consequence of the trpC mutation, may have contributed to the suppression of Pythium damping-off by the mutant.