Production of Purpureocillium lilacinum and Pochonia chlamydosporia by Submerged Liquid Fermentation and Bioactivity against Tetranychus urticae and Heterodera glycines through Seed Inoculation.

Pochoniachlamydosporia and Purpureocilliumlilacinum are fungal bioagents used for the sustainable management of plant parasitic nematodes. However, their production through submerged liquid fermentation and their use in seed treatment have been underexplored. Therefore, our goal was to assess the effect of different liquid media on the growth of 40 isolates of P. lilacinum and two of P. chlamydosporia. The most promising isolates tested were assessed for plant growth promotion and the control of the two-spotted spider mite (Tetranychus urticae) and the soybean cyst nematode (Heterodera glycines). Most isolates produced > 108 blastospores mL−1 and some isolates produced more than 104 microsclerotia mL−1. Microsclerotia of selected isolates were used to inoculate common bean (Phaseolus vulgaris L.) seeds in greenhouse trials. All fungal isolates reduced the T. urticae fecundity in inoculated plants through seed treatment, while P. chlamydosporia ESALQ5406 and P. lilacinum ESALQ2593 decreased cyst nematode population. Purpureocillium lilacinum was more frequently detected in soil, whereas P. chlamydosporia colonized all plant parts. Pochonia chlamydosporia ESALQ5406 improved the root development of bean plants. These findings demonstrate the possibility of producing submerged propagules of P. chlamydosporia and P. lilacinum by liquid culture, and greenhouse trials support the applicability of fungal microsclerotia in seed treatment to control P. vulgaris pests.

Slight induction and strong inhibition of Heterodera glycines hatching by short-chain molecules released by different plant species.

New management tools are necessary to reduce the damage caused by the soybean cyst nematode (SCN), Heterodera glycines. Identification of molecules that can stimulate second-stage juveniles (J2) hatching in an environment without food may contribute to that. In in vitro experiments, we evaluate the effect of volatile organic compounds (VOCs) released by soybean (Glycine max), bean (Phaseolus vulgaris), ryegrass (Lolium multiflorum), and alfalfa (Medicago sativa) on H. glycines egg hatching. VOCs released by all plant species significantly (p < 0.05) increased egg hatching. Short-chain molecules released by leaves and roots of soybean and bean increased the hatching up to 71.4%. The analysis of the volatilome done by gas chromatography coupled with mass spectrometry revealed 44 compounds in the plant emissions. Four of them, namely 3-octanol, 1-hexanol, hexanal and linalool were tested individually as hatching inductors. Under concentrations of 200, 600, and 1,000 µg/ml there was no hatching induction of H. glycines J2 by these compounds. On the other hand, in these concentrations, the compounds 3-octanol and 1-hexanol caused hatching reduction with values similar to the commercial nematicide carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methyl carbamate). In subsequent tests, the compounds 1-hexanol and 3-octanol showed lethal concentration values required to kill 50% of thenematode population (LC50) of 210 and 228 µg/ml, respectively, in the first experiment and, 230 and 124 µg/mlin the second one. Although we have not identified any molecules acting as hatching factor (HF), here we present a list (44 candidate molecules) that can be explored in future studies to find an efficient HF.

Evaluation of the Combined Effect of Heterodera glycines and Macrophomina phaseolina on Soybean Yield in Naturally Infested Fields with Spatial Regression Analysis and in Greenhouse Studies.

Heterodera glycines, the soybean cyst nematode, and Macrophomina phaseolina, causal agent of charcoal rot, are economically important soybean pathogens. The impact and effect of these pathogens on soybean yield in coinfested fields in the Midwest production region is not known. Both pathogens are soilborne, with spatially aggregated distribution and effects. Spatial regression analysis, therefore, is an appropriate method to account for the spatial dependency in either the dependent variable or regression error term from data produced in fields naturally infested with H. glycines and M. phaseolina. The objectives of this study were twofold: to evaluate the combined effect of H. glycines and M. phaseolina on soybean yield in naturally infested commercial fields with ordinary least squares and spatial regression models; and to evaluate, under environmentally controlled conditions, the combined effect of H. glycines and M. phaseolina through nematode reproduction and plant tissue fungal colonization. Six trials were conducted in fields naturally infested with H. glycines and M. phaseolina in Ohio. Systematic-grid sampling was used to determine the population densities of H. glycines and M. phaseolina, and soybean yield estimates. Though not used in any statistical analysis, M. phaseolina colony forming units from plant tissue, charcoal rot severity, and H. glycines type were also recorded and summarized. In two greenhouse experiments, treatments consisted of H. glycines alone, M. phaseolina alone, and coinfestation of soybean with both pathogens. Moran's I test indicated that the yield from five fields was spatially correlated (P < 0.05) and aggregated. In these fields, to account for spatial dependence, spatial regression models were fitted to the data. Spatial regression analyses revealed a significant interaction effect between H. glycines and M. phaseolina on soybean yield for fields with high initial population densities of both pathogens. In the greenhouse experiments, H. glycines reproduction was significantly (P < 0.05) reduced in the presence of M. phaseolina; however, soybean tissue fungal colonization was not affected by the presence of H. glycines. The direct mechanisms by which H. glycines and M. phaseolina interact were not demonstrated in this study. Future studies must be conducted in the field and greenhouse to better understand this interaction effect.

Effects of Tillage Practices on Dissemination and Spatial Patterns of Heterodera glycines and Soybean Yield.

Two field experiments were conducted in central Iowa to assess the effects of tillage on Heterodera glycines dissemination and reproduction and soybean (Glycine max) yield. Plots in both experiments were artificially infested with equivalent numbers of H. glycines cysts. In one experiment, plots were left noninfested or received aggregated or uniform infestation, and a susceptible soybean cultivar was grown for 3 years. By the end of the first growing season and through the second, H. glycines population densities were consistently greater (P ≤ 0.05) in uniformly infested plots than in plots with aggregated infestations. No differences in soybean yield among the treatments were detected. In a second experiment, a 1-m2 area of each plot was infested with H. glycines cysts, susceptible soybeans were grown for four seasons, and crop residue was managed with either ridge-, conventional-, reduced-, or no-tillage. After 1 year, nematode population densities were significantly (P ≤ 0.05) greater in conventional- and reduced-tillage treatments than in no- and ridge-tillage treatments. After 2 years, H. glycines had been disseminated 6.9 m from the infestation site in conventional- and reduced-tillage treatments but only 0.5 and 1.4 m for no-tillage and ridge-tillage treatments, respectively. After 3 years, H. glycines population densities were 10 times greater in conventional- and reduced-tillage treatments than in the no-tillage treatment; conventional-tillage was the only treatment with yield significantly lower (P ≤ 0.05) than the noninfested control. Aggregation of H. glycines eggs was greater (P ≤ 0.05) in no- and ridge-tillage treatments than in conventional- and reduced-tillage treatments. Results indicate tillage can quickly disseminate H. glycines in newly infested fields, facilitating more rapid nematode reproduction and subsequent yield loss.

Development of Heterodera glycines in resistant and susceptible soybean cultivars / Desenvolvimento de Heterodera glycines em cultivares resistentes e suscetível de soja

The objective of this study was to evaluate the development of race 3 of H. glycines in susceptible and resistant soybean cultivars. A completely randomized block design with four replications was used for the assessments, which involved Liderança, Centennial and Doko cultivars. Soybean plants with the first pair of leaves developed were inoculated with 4,000 eggs and evaluated 3, 6 and 10 days after the inoculation (DAI). The multiplication of the nematode was assessed by counting the numbers of nematodes per root system using a stereoscopic magnifying glass. The nematode was detected in all treatments but only second phase juveniles (J2) were found in the resistant cultivars Liderança and Centennial, in all evaluation phases. In the susceptible cultivar Doko, however, J2 juveniles in all evaluations and J3 and J4 10 DAI were observed. The results indicate that the nematode does not complete its cycle, which was interrupted around the 10th day after inoculation, in the resistant Liderança and Centennial cultivars.

O objetivo deste estudo foi avaliar o desenvolvimento da raça 3 de Heterodera glycines nas cultivares de soja Doko, Liderança e Centennial. O delineamento experimental foi inteiramente casualizado, com quatro repetições. Plantas de soja com o primeiro par de folhas desenvolvidas foram inoculadas com 4000 ovos e as avaliações foram realizadas 3, 6 e 10 dias após a inoculação (DAI). Avaliou-se o desenvolvimento do nematóide contando, sob lupa esteroscópica, o número de juvenis por sistema radicular. Nas cultivares resistentes Liderança e Centennial foram encontrados, em todas as épocas, somente juvenis de segundo estádio (J2). Já na cultivar suscetível Doko foi observada, além de J2 em todas as épocas, a presença de juvenis de terceiro (J3) e quarto (J4) estádios na avaliação 10 DAI. Assim, nas cultivares resistentes Liderança e Centennial o nematóide não completou seu ciclo que foi interrompido por volta do décimo DAI.

Development of Heterodera glycines in resistant and susceptible soybean cultivars / Desenvolvimento de Heterodera glycines em cultivares resistentes e suscetível de soja

The objective of this study was to evaluate the development of race 3 of H. glycines in susceptible and resistant soybean cultivars. A completely randomized block design with four replications was used for the assessments, which involved Liderança, Centennial and Doko cultivars. Soybean plants with the first pair of leaves developed were inoculated with 4,000 eggs and evaluated 3, 6 and 10 days after the inoculation (DAI). The multiplication of the nematode was assessed by counting the numbers of nematodes per root system using a stereoscopic magnifying glass. The nematode was detected in all treatments but only second phase juveniles (J2) were found in the resistant cultivars Liderança and Centennial, in all evaluation phases. In the susceptible cultivar Doko, however, J2 juveniles in all evaluations and J3 and J4 10 DAI were observed. The results indicate that the nematode does not complete its cycle, which was interrupted around the 10th day after inoculation, in the resistant Liderança and Centennial cultivars.

O objetivo deste estudo foi avaliar o desenvolvimento da raça 3 de Heterodera glycines nas cultivares de soja Doko, Liderança e Centennial. O delineamento experimental foi inteiramente casualizado, com quatro repetições. Plantas de soja com o primeiro par de folhas desenvolvidas foram inoculadas com 4000 ovos e as avaliações foram realizadas 3, 6 e 10 dias após a inoculação (DAI). Avaliou-se o desenvolvimento do nematóide contando, sob lupa esteroscópica, o número de juvenis por sistema radicular. Nas cultivares resistentes Liderança e Centennial foram encontrados, em todas as épocas, somente juvenis de segundo estádio (J2). Já na cultivar suscetível Doko foi observada, além de J2 em todas as épocas, a presença de juvenis de terceiro (J3) e quarto (J4) estádios na avaliação 10 DAI. Assim, nas cultivares resistentes Liderança e Centennial o nematóide não completou seu ciclo que foi interrompido por volta do décimo DAI.