Isolation and activity of Xenorhabdus antimicrobial compounds against the plant pathogens Erwinia amylovora and Phytophthora nicotianae.

AIMS: Broad-spectrum antibiotics produced by symbiotic bacteria [entomopathogenic bacterium (EPB)] of entomopathogenic nematodes keep monoxenic conditions in insect cadavers in soil. This study evaluated antibiotics produced by EPB for their potential to control plant pathogenic bacteria and oomycetes. METHODS AND RESULTS: Entomopathogenic bacterium produce antibiotics effective against the fire blight bacterium Erwinia amylovora, including streptomycin resistant strains, and were as effective in phytotron experiments as kasugamycin or streptomycin. Xenorhabdus budapestensis and X. szentirmaii antibiotics inhibited colony formation and mycelial growth of Phytophthora nicotianae. From X. budapestensis, an arginine-rich fraction (bicornutin) was adsorbed by Amberlite((R)) XAD 1180, and eluted with methanol : 1 n HCI (99 : 1). Bicornutin inactivated zoospores, and inhibited germination and colony formation of cystospores at <<25 ppm. An UV-active molecule (bicornutin-A, MW = 826), separated by HPLC and thin-layer chromatography, was identified as a novel hexa-peptide : RLRRRX. CONCLUSIONS: Xenorhabdus budapestensis produces metabolites with strong antibacterial and cytotoxic activity. Individual compounds can be isolated, identified and patented, but their full antimicrobial potential may be multiplied by synergic interactions. SIGNIFICANCE AND IMPACT OF THE STUDY: Active compounds of two new Xenorhabdus species might control plant diseases caused by pathogens of great importance to agriculture such as Erw. amylovora and P. nicotianae.

First Report of Phytophthora citricola on False Cypress in Hungary.

In May 2005, an estimated 10 to 15% mortality of various cultivars of false cypress (also named Lawson cypress or Port-Orford-cedar [Chamaecyparis lawsoniana]) with severe wilt was observed in field stands of an ornamental nursery in western Hungary. Wilted plants had rot-associated reduction of their root system. Root discoloration and occasional chlorosis of lower leaves commenced on potted 3-year-old plants that were held in the open air for 10 to 12 months before planting. Four species of Phytophthora (P. lateralis, P. eriugena, P. hibernalis, and P. cinnamomi) have been reported on this host (2). Direct plating of discolored roots from the most susceptible cultivar (Silver Globus) onto a selective potato dextrose agar or carrot agar medium yielded pure cultures that developed white, stellate colonies with sparse aerial mycelia. The hyphal growth was optimal at 25°C, but the growth above 32°C and below 4°C was completely inhibited. Single, terminal sporangia on simple (occasionally sympodial) sporangiophores formed abundantly in nonsterile soil filtrate but not in agar. Sporangia, 31 to 67 µm (59.1 ± 9.3 µm) long and 25 to 39 µm (31.5 ± 4.0 µm) wide, were noncaducous and semipapillate, variable in shape, mostly obpyriform, rarely obovoid, ovoid-ellipsoid and spherical or bifurcated and distorted, and the exit pore was narrow (7.2 ± 0.8 µm). No external or internal proliferation and no hyphal swellings or chlamydospores were observed. The isolates were homothallic with smooth-walled oogonia (27.3 ± 3.4 µm in diameter) and paragynous antheridia. The oospores (24.7 ± 2.1 µm in diameter) were plerotic. The morpho-physiological features suggested that our isolates belonged to Waterhouse's Group III, and in particular, represented P. citricola. This was confirmed by cellulose acetate electrophoresis of malate dehydrogenase; the isozyme pattern of false cypress isolate was identical to that of the ITS-sequenced (NCBI Accession No. AY366193) P. citricola isolate from a Hungarian alder forest (1). Pathogenicity tests on four 3-year-old potted false cypress (cv. Silver Globus) plants in the greenhouse resulted in rapidly developing (within 2 weeks) sunken, necrotic lesions at the stem base around the site of wound inoculation with a 5-mm-diameter mycelial agar plug. After 12 weeks, each inoculated plant wilted and died. The causal agent was consistently reisolated from necrotic tissues. In Hungary, P. citricola was first isolated and identified from alder forest soil (1). Nonetheless that false cypress has been listed as the host of P. citricola in Norway and Poland (3,4), to our knowledge, this report is the first definitive description of this Phytophthora sp. on this host. References: (1) J. Bakonyi et al. Plant Pathol. 52:807, 2003. (2) D. C. Erwin and O. K. Ribeiro. Pages 282-287 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) V. Talgø V. and A. Stensvand. Grønn kunnskap e 7(101G):1, 2003. (4) K. Wiejacha et al. Page 45 in: Improvement and Unification of Plant Disease Diagnostics. Abstracts of International Workshop, Skierniewice, Poland, 2004.

First Report of Phytophthora Root and Collar Rot of Alder in Hungary.

In June 1999, a disease associated with mortality of Alnus glutinosa, was observed in a 12- to 18-year-old peatland plantation in northwest Hungary. The root and collar rot symptoms were similar to those caused by Phytophthora cambivora in tree species other than alders. Nine isolations were made from diseased roots and soil samples using the Rhododendron leaf baiting method. Three isolates recovered from two sites, approximately 2 km apart, exhibited similar growth and morphology in vitro and were pathogenic to 2-year-old trees of A. glutinosa following inoculation of root collars. All three isolates had amphigynous long, two-celled antheridia. The mean diameter of oogonia ranged from 39.5 to 64.6 µm. They also produced nonpapillate, ellipsoid, non-caducous sporangia 26.9 to 50.5 µm long and 19.3 to 38.5 µm wide with broad exit pores in soil filtrate. These characteristics were similar to those reported for Phytophthora on alder from elsewhere in Europe and for P. cambivora that is not a pathogen of alder (1,2). However, Hungarian isolates from alder, in contrast to P. cambivora, were homothallic like previously recorded isolates from alder, formed nonornamented oogonia and developed colonies at lower optimum (approximately 25°C) and maximum (approximately 30°C) temperatures on carrot agar. A comparison with Phytophthora from alder from other countries (courtesy of C. M. Brasier) showed that the Hungarian isolates have smooth-walled oogonia typical of Swedish isolates rather than the ornamented oogonia of U.K. isolates, but have the appressed, slightly woolly colony morphology like U.K. isolates rather than the fluffy growth found in Swedish isolates. Moreover, cellulose acetate electrophoresis of glucose-6-phosphate isomerase revealed one homodimer band in Hungarian isolates that was identical with that of the Swedish isolate from alder P876 and isolates P1010 and P1011 of P. cambivora (courtesy of C. M. Brasier). This band comigrated with the middle one of the five-banded U.K. standard isolate P772. Molecular evidence (2) indicates that the Phytophthora from alder with its unusual characteristics is not a species in the strict sense but comprises natural hybrids that may have originated in an interspecific hybridization event between a P. cambivora-like species and an unknown species similar to P. fragariae. On this basis, the Hungarian Phytophthora from alder might have evolved similarly. It remains to be determined whether the pathogen was introduced or has developed independently. References: (1) C. M. Brasier et al. Plant Pathol. 44:999, 1995. (2) C. M. Brasier et al. Proc. Natl. Acad. Sci. USA 96:5878, 1999.

First Report of the A2 Mating Type of Phytophthora infestans on Potato in Hungary.

Severe symptoms of potato late blight were observed in July 1996 on potato (Solanum tuberosum L.) cv. Desirée grown on a farm in western Hungary. Isolation was made directly from diseased leaf tissues onto selective pea-agar medium. A recovered isolate, H2a, was identified as Phytophthora infestans (Mont.) de Bary on the basis of Koch's postulates and morphological characteristics of the fungus. Pairing of H2a with isolates of known mating types, A1 and A2 from Germany, revealed that it represents the A2 mating type. After a 2-week incubation on pea-agar medium at 20°C, oospores formed in abundance in the region of contact between the colonies of H2a and the A1 mating type isolate. After extended incubation scattered formation of gametangia was observed when isolate H2a had been paired with itself or with the A2 mating type isolate. The same phenomenon of presumed self fertilization also took place when single, zoospore-derived colonies of H2a were combined with one another or with the known A2 isolate. Of an incomplete set of potato differentials, leaves of potato genotypes r, R2, R3, R4, R1.2.3.4, R7, R8, and R11 were all susceptible to infection with a zoospore suspension of the isolate H2a. The complex virulence phenotype of H2a, its tolerance to metalaxyl in agar cultures and on leaf disks (EC50 >100 mg liter-1), and its A2 mating type behavior collectively suggest that H2a represents a genotype that recently has been introduced into Hungary.

PCR amplification of species-specific DNA sequences can distinguish among Phytophthora species.

We used PCR to differentiate species in the genus Phytophthora, which contains a group of devastating plant pathogenic fungi. We focused on Phytophthora parasitica, a species that can infect solanaceous plants such as tomato, and on Phytophthora citrophthora, which is primarily a citrus pathogen. Oligonucleotide primers were derived from sequences of a 1,300-bp P. parasitica-specific DNA segment and of an 800-bp P. citrophthora-specific segment. Under optimal conditions, the primers developed for P. parasitica specifically amplified a 1,000-bp sequence of DNA from isolates of P. parasitica. Primers for P. citrophthora similarly and specifically amplified a 650-bp sequence of DNA from isolates of P. citrophthora. Detectable amplification of these specific DNA sequences required picogram quantities of chromosomal DNA. Neither pair of primers amplified these sequences with DNAs from other species of Phytophthora or from the related genus Pythium. DNAs from P. parasitica and P. citrophthora growing in infected tomato stem tissue were amplified as distinctly as DNAs from axenic cultures of each fungal species. This is the first report on PCR-driven amplification with Phytophthora species-specific primers.

Compatible and incompatible rhizobia alter membrane potentials of soybean root cells.

Inoculation with Rhizobium japonicum or R. meliloti reduced the electrical transmembrane potential (E(m)) of soybean (Glycine max [L.] Merr.) root cells within 1 day. The response could be attributed to altered diffusion potential (E(D)). E(m) values return to control levels by the second day after inoculation, but again were reduced in R. meliloti-inoculated tissue on the seventh day. Increased concentrations of sodium phosphate in the perfusion solution magnified the effects of inoculation on E(m). Neither heat-killed rhizobia nor living cells of Pseudomonas fluorescens elicited the response. The E(m) and E(D) of nodule cells were nearly 20% lower than corresponding values from adjacent cortical cells of the root.