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The fungus Leptolegnia chapmanii is highly pathogenic to mosquito larvae in Argentina. We studied if physical and chemical characteristics of the water from mosquito breeding sites affect pathogenicity, and the infectivity of zoospores of L. chapmanii. Water samples were taken from pools filled by rains, urban ditches with domestic waste water, pools filled by overflow from Río de la Plata, and flower vases from the Cemetery of La Plata city. Sub-samples of water were analyzed for physical and chemical characteristics, while other sub-samples were used for laboratory bioassays. Containers with 150 ml of water samples, 25 Aedes aegypti larvae, and 2.8 x 10(5) zoospores of L. chapmanii, were incubated under controlled environment, and larval mortality was recorded after 48 h. There were highly significant differences among mortalities in water from cemetery vases (70.2%), rain pools water (99.5%), and pools with water from Río de la Plata (95%). There were no significant differences among larval mortalities in water from ditches, rain pools and Río de la Plata pools. Leptolegnia chapmanii was successful as a biological control agent in all kinds of tested water qualities, producing high larval mortality.

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 degrees C but not at 5 or 40 degrees C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 degrees C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 degrees C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.

The effects of water volume, container surface area and the density of hosts and fungal zoospores on the infectivity of the oomycete fungus Leptolegnia chapmanii to Aedes aegypti were investigated in the laboratory. Late 3rd or early 4th instars from a laboratory colony were used as hosts in all assays. Fourth instars infected with L. chapmanii for 48 h (6.1 +/- 0.2 x 10(4) zoospores/larva) were used as inoculum. Mortality rates were >90% in containers with 20 or fewer larvae when exposed to 1 infected larva, but they decreased to 37% in containers with 60 larvae. Mortality rates varied from 82% to 92% when healthy 4th instars were exposed to 1 and 2 infected larvae, respectively, whereas 100% mortality was obtained with > or =3 infected larvae. Infection and mortality rates in containers with volumes that varied from 300 to 5,000 ml but that had similar water surface area (397 cm2) varied from 89 to 92%, respectively. When water volume was held constant at 250 ml in containers with variable surface areas (14.5-875 cm(2)), larval mortality varied from 96% to 25%, respectively. The ability of L. chapmanii to infect mosquito larvae is dose dependent and influenced by larval density and surface area.

ABSTRACT Isolates of the Diaporthe/Phomopsis (D/P) complex were collected in the main soybean producing area of Argentina during the 1996-97, 1997-98, and 1998-99 growing seasons. Twenty-three morphologic characters related to type of colonies, stroma, pycnidia and conidia, presence of perithecia, and asci length were studied by principal component analysis (PCA). Genomic DNA were analyzed by the random amplified polymorphic DNA (RAPD) technique. From both studies, 18 isolates were identified as D/P complex and grouped in four major taxa: (i) Diaporthe phaseolorum var. meridionalis, (ii) D. phaseolorum var. caulivora, (iii) D. phaseolorum var. sojae, and (iv) Phomopsis longicolla. In addition to distinguishing interspecific and intraspecific variability, molecular markers allowed the detection of differences among isolates within the same variety. Pathogenicity was assayed in the greenhouse, by the toothpick method, inoculating the D/P isolates to soybean genotypes carrying different resistance genes (Rdc1, Rdc2, Rdc3, and Rdc4) against soybean stem canker (SSC). Pathogenic analysis distinguished two main groups: (i) the SSC-producing isolates, including D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora, and (ii) the non-SSC-producing isolates, including D. phaseolorum var. sojae and P. longicolla. Cultivar RA-702 (susceptible control) was compatible with both D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora isolates; meanwhile, Tracy-M (Rdc1 and Rdc 2 genes) was incompatible with D. phaseolorum var. meridionalis but compatible with D. phaseolorum var. caulivora isolates. The fact that Rdc1 and Rdc2 together (as in Tracy-M) confer an almost immune reaction to all assayed isolates of D. phaseolorum var. meridionalis but were ineffective against the D. phaseolorum var. caulivora isolates evaluated suggests that the virulence or avirulence genes in D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora are different. Moreover, physiological races of D. phaseolorum var. meridionalis were detected by using differential soybean genotypes carrying distinct single Rdc genes. As far as we know, this is the first report on the existence of physiological races of D. phaseolorum var. meridionalis in South America. Selective pressure due to deployment of resistant host cultivars may have changed the frequency of the virulence or avirulence genes within the population of D. phaseolorum var. meridionalis. On the whole, our results show that pathogenic variability of D. phaseolorum in the core soybean-producing area of Argentina is higher than previously recognized.

A soybean stem canker (SSC) outbreak caused by Diaporthe phaseolorum (Cooke & Ellis) Sacc. var. meridionalis Fernández was reported in Santa Fe, Argentina, in 1997 (3). In 1999 an isolate, which was morphologically distinct from D. phaseolorum var. meridionalis, was obtained from stems of field-grown soybean plants exhibiting SSC symptoms, at Oliveros, Santa Fe, Argentina (Lat. 32° 33'S, Lon. 60° 51'W). Disease incidence was 76% in the field where samples were collected. The pathogen was isolated in darkness at 25°C on potatoglucose agar acidified with 0.2% lactic acid (3). The isolate produced white colonies with compact and tufted mycelium that changed to yellow and light tan with age. Stromata and pycnidia were not produced. After 35 days in culture, clustered perithecia were frequently observed on stem segments. Fifty asci, five from each of 10 perithecia, and bicellular, biguttulated ascospores were measured. Ascus mean length was 26.9 ± 2.5 μm and width was 5.3 ± 0.5 μm; ascospore mean length was 8.3 ± 0.6 μm and width was 2.6 ± 0.1μm. Based on these features, the new isolate was classified as D. phaseolorum var. caulivora Athou & Caldwell (1). To further compare the new isolate with previous identified ones, a principal component analysis (PCA, SAS Systems) was performed using seven isolates of D. phaseolorum var. meridionalis, three isolates of D. phaseolorum var. sojae, and two isolates of Phomopsis longicolla. Seventeen morphological characters, all related with the color and texture of the colonies, the presence and shape of the pycnidia and conidia, the presence and type of stromata and perithecia, and the length of the asci, were compared. According to the PCA analysis, the principal characters that discriminated SSC producing isolates (D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora) from non-SSC producing ones (D. phaseolorum var. sojae and P. longicolla) were the development of perithecia (r = 0.98) and low frequency stromata (r = 0.98) in D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora isolates. The principal components that discriminated SSC producing isolates were the more compact and tufted aspect of the mycelia (r = 0.95) and the shorter length of the asci (r = 0.83) in D. phaseolorum var. caulivora compared with D. phaseolorum var. meridionalis. Pathogenicity trials were performed under greenhouse conditions by inoculating D. phaseolorum var. caulivora mycelia in hypocotyls of soybean seedlings by the toothpick method (2). Typical SSC symptoms were observed on susceptible plants and the pathogen was re-isolated and identified from stem portions of the first internode above the inoculation point. Pathogenicity trials were repeated twice with similar results. This is the first report of D. phaseolorum var. caulivora in Argentina and, as far as we know, in all of South America. References: (1) F. A. Fernández et al. 1999. Stem canker. Pages 32-35 in: Compendium of Soybean Diseases, 4th ed. APS Press, St. Paul, MN. (2) B. L. Keeling. Phytopathology 72:807-809, 1982. (3) R. N. Pioli et al. Plant Dis. 81:1215, 1997.