Antifungal activity of Xenorhabdus spp. and Photorhabdus spp. against the soybean pathogenic Sclerotinia sclerotiorum.

The fungus, Sclerotinia sclerotiorum, causes white mold disease and infects a broad spectrum of host plants (> 500), including soybean with yield losses of up to 70%. Biological control is a potential alternative for management of this severe plant pathogen, and relative to chemical fungicides, provides broad benefits to the environment, farmers and consumers. The symbiotic bacteria of entomopathogenic nematodes, Xenorhabdus spp. and Photorhabdus spp., are characterized by the production of antimicrobial compounds, which could serve as potential sources for new bio-fungicides. The objectives of this study were to assess cell-free supernatants (CFS) of 16 strains of these bacteria cultures on S. sclerotiorum mycelium growth; assess the volatiles of X. szentirmaii cultures on the fungus mycelium and sclerotium inhibition; and evaluate the X. szentirmaii cultures as well as their CFS on the protection of soybean seeds against the white mold disease. Among the 16 strains, the CFS of X. szentirmaii showed the highest fungicidal effect on growth of S. sclerotiorum. The CFS of X. szentirmaii inhibited > 98% of fungus growth from mycelium and sclerotia, whereas the volatiles generated by the bacterium culture inhibited to 100% of fungus growth and 100% of sclerotia production. The bacterial culture diluted to 33% in water and coated on soybean seeds inhibited S. sclerotiorum and protected soybean plants, allowing 78.3% of seed germination and 56.6% of plant development. Our findings indicate potential for a safe and novel control method for S. sclerotiorum in soybean. Moreover, this is the first study to indicate that volatile organic compounds from Xenorhabdus spp. can be used in plant disease suppression.

Trans-cinnamic acid and Xenorhabdus szentirmaii metabolites synergize the potency of some commercial fungicides.

Development of novel approaches for the control of fungal phytopathogens is desirable. In this study we hypothesized that the combination of commercial fungicides with certain enhancing agents could result in synergistic levels of control. Prior research has indicated that trans-cinnamic-acid (TCA), a metabolite of the bacteria Photorhabdus luminescens and metabolites of Xenorhabdus szentirmaii are particularly toxic to various phytpathogenic fungi when compared to metabolites of other Xenorhabdus or Photorhabdus spp. In this study we explored the efficacy of commercial fungicide interactions when combined with either TCA or X. szentirmaii. Fungicides (active ingredient) included Abound® (Azoxystrobin), Serenade® (Bacillus subtilis), Elast® (dodine), Regalia® (extract of Reynoutria sachalinensis), Prophyt® (potassium phosphite) and PropiMax® (propiconazole). In laboratory experiments, singly-applied or combined agents were assessed for fungicidal activity against four plant-pathogenic fungi, Monilinia fructicola, Rhizoctonia solani, Colletotrichum gloeosporioides and Fusarium oxysporum. Fungicidal activity was measured by the phytopathogen's growth on potato dextrose agar with and without fungicide. The interactions between fungicidal agents were determined as antagonistic, additive or synergistic. For suppression of M. fructicola, synergy was observed between TCA when combined with certain concentrations of Elast®, PropiMax®, Regalia®, Prophyte® or Serenade®, and for combinations of X. szentirmaii with Abound®. For suppression of R. solani, synergy was observed between TCA combined with Regalia® or Serenade®. Additionally, when TCA was combined with X. szentirmaii synergistic levels of suppression to M. fructicola were observed. Other combinations of TCA or X. szentirmaii with the fungicides or using alternate concentrations were either additive or occasionally antagonistic in nature. Our results indicate that TCA and X. szentirmaii can each act as strong synergists to enhance fungicidal efficacy. These results may be used to reduce negative environmental impacts of pesticide use while improving control of plant diseases. Additional research is needed to explore the diversity of the synergistic effects and confirm our observations under field conditions.

The Effects of Nutrient Concentration, Addition of Thickeners, and Agitation Speed on Liquid Fermentation of Steinernema feltiae.

Entomopathogenic nematode production in liquid fermentation still requires improvements to maximize efficiency, yield, and nematode quality. Therefore, this study was aimed at developing a more suitable liquid medium for mass production of Steinernema feltiae, by assessing the effects of nutrient concentration, thickeners (primarily agar), and agitation speed on infective juvenile (IJ) yield. Base medium (BM) contained yeast extract (2.3%), egg yolk (1.25%), NaCl (0.5%), and corn oil (4%). All media were inoculated with Xenorhabdus bovienii, and 2 d later, with 2-d-old S. feltiae juveniles. For the nutrient concentration experiment, we evaluated the base medium versus a modified base medium containing all the components, but with 3× concentrations of yeast extract (6.9%), egg yolk (3.75%), and corn oil (12%). The nematodes and bacteria were cultured in 150-ml Erlenmeyer flasks containing 50 ml of liquid medium at (25°C) and 180 rpm on a rotary shaker incubator. To assess the effect of thickeners, IJs were inoculated in BM with agar (0.2%), carrageen (0.2%), and carboxymethyl cellulose (0.2% and 0.5%). The addition of 3× more nutrients relative to the BM resulted in a significantly lower yield of nematodes. For agar and agitation speed experiments, five levels of agar in the BM (0%, 0.2%, 0.4%, 0.6%, and 0.8% agar) and two agitation speeds (180 and 280 rpm) were evaluated for production. Increasing agitation speed from 180 to 280 rpm and higher levels of agar in the medium (> 0.2%) significantly increased the yield of bacteria. At the lower agitation speed, media amended with 0.4% and 0.6% agar produced higher nematode yields compared to media without agar. Media with 0.2% and 0.8% agar resulted in intermediate levels of nematode production. At the higher agitation speed, media supplemented with 0.8% agar resulted in the lowest yield of nematodes when compared to the other media tested. Results indicated that increasing nutrient concentration levels was detrimental to nematode production. Also, media containing agar (0.4% and 0.6%) increased nematode yields when cultures were grown at low agitation speed. When IJs were used as the inoculum, 0.2% agar also enhanced recovery and nematode yield at the higher agitation speed.

Multifaceted effects of host plants on entomopathogenic nematodes.

The success of parasites can be impacted by multi-trophic interactions. Tritrophic interactions have been observed in parasite-herbivore-host plant systems. Here we investigate aspects of multi-trophic interactions in a system involving an entomopathogenic nematode (EPN), its insect host, and host plant. Novel issues investigated include the impact of tritrophic interactions on nematode foraging behavior, the ability of EPNs to overcome negative tritrophic effects through genetic selection, and interactions with a fourth trophic level (nematode predators). We tested infectivity of the nematode, Steinernema riobrave, to corn earworm larvae (Helicoverpa zea) in three host plants, tobacco, eggplant and tomato. Tobacco reduced nematode virulence and reproduction relative to tomato and eggplant. However, successive selection (5 passages) overcame the deficiency; selected nematodes no longer exhibited reductions in phenotypic traits. Despite the loss in virulence and reproduction nematodes, first passage S. riobrave was more attracted to frass from insects fed tobacco than insects fed on other host plants. Therefore, we hypothesized the reduced virulence and reproduction in S. riobrave infecting tobacco fed insects would be based on a self-medicating tradeoff, such as deterring predation. We tested this hypothesis by assessing predatory success of the mite Sancassania polyphyllae and the springtail Sinella curviseta on nematodes reared on tobacco-fed larvae versus those fed on greater wax moth, Galleria mellonella, tomato fed larvae, or eggplant fed larvae. No advantage was observed in nematodes derived from tobacco fed larvae. In conclusion, our results indicated that insect-host plant diet has an important effect on nematode foraging, infectivity and reproduction. However, negative host plant effects, might be overcome through directed selection. We propose that host plant species should be considered when designing biocontrol programs using EPNs.

Steinernema brazilense n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Mato Grosso, Brazil.

A new entomopathogenic nematode, Steinernema brazilense n. sp., was isolated from a single soil sample collected from a natural forest in Mato Grosso do Sul state, Brazil. S. brazilense n. sp. is characterized morphologically by features of infective juveniles (IJ), males and females. For the IJ, body length averaging 1157 (1023-1284)microm, distance from anterior end to excretory pore 95 (87-102)microm, from anterior end to end of esophagus 148 (139-153)microm, tail length 85 (80-104)microm, D% and E% values 63 (58-70) and 106 (95-118.0), respectively. Lateral field pattern variable; the formula for the arrangement of ridges from head to tail is: 2, 4, 6, 8, 6, 2. For the male, the diagnostic characters include spicule averaging 83 (75-89)microm; D% about 65; the ratio SW% about 192. The length of spicule head is greater than width. Lateral field with one narrow ridge. First generation females are characterized by the presence of a ventral postanal swelling. S. brazilense n. sp. is morphologically close to Steinernema diaprepesi. It can be differentiated from S.diaprepesi by its longer IJ body length (1157 vs 1002microm), longer distance from anterior end to excretory pore (110 vs 75microm), a longer tail length (103 vs 83microm); males of the new species with longer spicule (83 vs 79microm). The new species can be distinguished further from other members of Steinernema glaseri group by characteristics of rDNA of ITS and D2D3 regions.

[First record of Zoophthora radicans (Entomophthorales: Entomophthoraceae) on adults of the Paraguay tea psyllid, Gyropsylla spegazziniana Lizer & Trelles (Hemiptera: Psyllidae), in Brazil]. / Primeiro Registro de Zoophthora radicans (Entomophthorales: Entomophthoraceae) em Adultos da Ampola-da-Erva-Mate, Gyropsylla spegazziniana Lizer & Trelles (Hemiptera: Psyllidae), no Brasil.

We report the first occurrence of Zoophthora radicans infecting adults of Gyropsylla spegazziniana Lizer & Trelles in a commercial Paraguay tea plantation (Ilex paraguariensis), in Cascavel, PR, Brazil. The fungus prevalence was high (90% of mortality), considered a natural epizooty.

Primeiro Registro de Zoophthora radicans (Entomophthorales: Entomophthoraceae) em Adultos da Ampola-da-Erva-Mate, Gyropsylla spegazziniana Lizer & Trelles (Hemiptera: Psyllidae), no Brasil / First Record of Zoophthora radicans (Entomophthorales: Entomophthoraceae) on Adults of the Paraguay Tea Psyllid, Gyropsylla spegazziniana Lizer & Trelles (Hemiptera: Psyllidae), in Brazil

A primeira ocorrência de Zoophthora radicans (Entomophthorales: Entomophthoraceae) infectando adultos de Gyropsylla spegazziniana Lizer & Trelles (Hemiptera: Psyllidae) em plantio comercial de erva-mate (Ilex paraguariensis), em Cascavel, PR, Brasil, é relatada. A prevalência do fungo foi elevada (90 por cento de mortalidade), sendo considerada uma epizootia natural.

We report the first occurrence of Zoophthora radicans infecting adults of Gyropsylla spegazziniana Lizer & Trelles in a commercial Paraguay tea plantation (Ilex paraguariensis), in Cascavel, PR, Brazil. The fungus prevalence was high (90 percent of mortality), considered a natural epizooty.

Application technology for the entomopathogenic nematodes Heterorhabditis indica and Steinernema sp. (Rhabditida: Heterorhabditidae and Steinernematidae) to control Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) in corn.

The effects of different application technologies were evaluated on the concentration, viability, and efficiency of infective juveniles of the nematodes Heterorhabditis indica Poinar, Karunakar & David and Steinernema sp. (IBCB-n6) to control Spodoptera frugiperda Smith on corn plants. Two hundred and eighty infective juveniles of Steinernema sp. were required to kill 100% third-instar fall armyworms in petri dishes, as compared to 400 infective juveniles of the H. indica nematode to obtain 75% fall armyworm control. It is possible to spray entomopathogenic nematodes without significant loss in their concentration and viability, with equipment that produces electrical charges to the spraying mix, and with those using hydraulic and rotary nozzle tips. The concentrations of infective juveniles of H. indica and Steinernema sp. nematodes were reduced by 28% and 53%, respectively, when hydraulic spraying nozzles that require 100-mesh filtrating elements were used. Tensoactive agents of the organosilicone and ethoxylate groups did not affect the viability of infective juveniles of Steinernema sp. juveniles. Spraying corn plants (V6 growth stage) with up to 288 million infective juveniles of Steinernema sp. per hectare, diluted in the spraying mix up to 800 L ha-1, with 0.01% ethoxylate tensoactive agent, or at the same volume followed by artificial rain (6 mm water depth) was not sufficient to control S. frugiperda in a controlled environment.

Application technology for the entomopathogenic nematodes Heterorhabditis indica and Steinernema sp. (Rhabditida: Heterorhabditidae and Steinernematidae) to control Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) in corn / Tecnologia de aplicação para os nematóides entomopatogênicos Heterorhabditis indica e Steinernema sp. (Rhabditida: Heterorhabditidae e Steinernematidae) para controle de Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae) na cultura do milho

The effects of different application technologies were evaluated on the concentration, viability, and efficiency of infective juveniles of the nematodes Heterorhabditis indica Poinar, Karunakar & David and Steinernema sp. (IBCB-n6) to control Spodoptera frugiperda Smith on corn plants. Two hundred and eighty infective juveniles of Steinernema sp. were required to kill 100 percent third-instar fall armyworms in petri dishes, as compared to 400 infective juveniles of the H. indica nematode to obtain 75 percent fall armyworm control. It is possible to spray entomopathogenic nematodes without significant loss in their concentration and viability, with equipment that produces electrical charges to the spraying mix, and with those using hydraulic and rotary nozzle tips. The concentrations of infective juveniles of H. indica and Steinernema sp. nematodes were reduced by 28 percent and 53 percent, respectively, when hydraulic spraying nozzles that require 100-mesh filtrating elements were used. Tensoactive agents of the organosilicone and ethoxylate groups did not affect the viability of infective juveniles of Steinernema sp. juveniles. Spraying corn plants (V6 growth stage) with up to 288 million infective juveniles of Steinernema sp. per hectare, diluted in the spraying mix up to 800 L ha-1, with 0.01 percent ethoxylate tensoactive agent, or at the same volume followed by artificial rain (6 mm water depth) was not sufficient to control S. frugiperda in a controlled environment.

O efeito de diversas tecnologias de aplicação foi avaliado sobre a concentração, viabilidade e eficácia dos juvenis infectantes dos nematóides Heterorhabditis indica Poinar, Karunakar & David (IBCB-n5) e Steinernema sp. (IBCB-n6) no controle da lagarta-do-cartucho Spodoptera frugiperda Smith na cultura do milho. Para o controle da lagarta-do-cartucho no terceiro estádio em placas de Petri foram necessários 280 juvenis infectantes de Steinernema sp., enquanto que 400 juvenis infectantes de H. indica controlaram apenas 75 por cento das lagartas. Podem-se pulverizar os entomopatógenos, sem que haja perda significativa na sua concentração e viabilidade, com equipamentos que forneçam carga elétrica à calda, ponta centrífuga e pontas hidráulicas. Entretanto, o emprego de pulverizadores com pontas que requerem elementos filtrantes com malha igual a 100 resultou em decréscimo na concentração de juvenis infectantes de H. indica e Steinernema sp., de 28 por cento e 53 por cento, respectivamente. Os tensoativos organosiliconado e etoxilados não afetaram a viabilidade dos juvenis infectantes de Steinernema sp. Nos experimentos de pulverização em plantas de milho (V6) com Steinernema sp., doses equivalentes a até 288 milhões de juvenis infectantes por hectare, diluídos em volume de calda de até 800 L ha-1 com 0,01 por cento do tensoativo etoxilado, ou nesse volume seguido de exposição a chuva artificial (lâmina de água de 6 mm), não foram suficientes para o controle de S. frugiperda em casa-de-vegetação.

Screening of entomopathogenic nematodes (Nemata: Rhabditida) and the efficiency of Heterorhabditis sp. against the sugarcane root spittlebug Mahanarva fimbriolata (Fabr.) (Hemiptera: Cercopidae)

Este estudo teve por objetivo avaliar a patogenicidade de seis nematóides contra ninfas da cigarrinha da raiz da cana-de-açúcar Mahanarva fimbriolata (Fabr.), em condições de laboratório, e a eficiência do mais virulento no controle do inseto em condições de campo. No laboratório, foram avaliados sete tratamentos representados por dois espécimes do gênero Heterorhabditis, três de Steinernema, um espécime de Steinernema glaseri (Steiner),e a testemunha. No campo foram realizados dois experimentos. No primeiro foram avaliados nove tratamentos: Heterorhabditis sp. (CB-n5) aplicado sobre a palhada nas doses de 3,3 x 109, 6,6 x 108, 3,3 x 108 e 6,6 x 107 juvenis infectivos (JI)/ha; os nematóides aplicados no solo (embaixo da palhada) nas mesmas quatro doses; e a testemunha. No segundo experimento foram testados quatro tratamentos: Heterorhabditis sp. (3,3 x 108 JI/ha); Metarhizium anisopliae (2,6 x 1012 conídios viáveis/ha); tiametoxam (Actara 200 WG) (1 kg/ha); e a testemunha. Em laboratório, Heterorhabditis sp. (CB-n5), Steinernema sp. (CB-n6) e Heterorhabditis sp. (CCA) foram os mais patogênicos, causando mortalidades de 100%, 98% e 96%, respectivamente. No primeiro experimento de campo, o nematóide Heterorhabditis sp. proporcionou até 70% de controle da cigarrinha, não havendo diferença significativa entre as doses e quanto à aplicação sobre a palhada ou sobre o solo. No segundo experimento, o inseticida proporcionou 67% de controle, não diferenciando significativamente do nematóide (56%) e do fungo (44%), em avaliação realizada sete dias após aplicação.

The pathogenicity of six entomopathogenic nematodes was assessed against nymphs of the sugarcane root spittlebug Mahanarva fimbriolata (Fabr.), in the laboratory. The efficiency of the most virulent agent was tested in the field. Seven laboratory treatments were designed with two specimens of the genus Heterorhabditis, three of Steinernema, one specimen of Steinernema glaseri (Steiner), and acontrol group. In the field, two experiments were conducted. The first experiment involved nine treatments:the control, four treatments with Heterorhabditis sp. (CB-n5) applied to the straw mulch at doses 6.6 x 107, 3.3 x 108, 6.6 x 108 and 3.3 x 109 IJs/ha, and four treatments with the nematodes applied to the soil and beneath the straw mulch layer, at the same doses. The second experiment contained four treatments: the control and applications of Heterorhabditis sp. (3.3 x 108 IJs/ha), the fungus Metarhizium anisopliae (2.6 x 1012 viable conidia/ha), and the chemical insecticide thiamethoxan (Actara 200 WG) (1 kg/ha). Heterorhabditis sp. (CB-n5), Steinernema sp. (CB-n6), and Heterorhabditis sp. (CCA) were the threemost virulent nematodes (100%, 98%, and 96% mortality, respectively). In the first field experiment, Heterorhabditis sp. controlled 74% of the insects, with no significant difference among doses or between application to the soil or mulch. In the second experiment, the chemical insecticide provided 67% control, not differing statistically from nematode (56%) or fungus (44%) control, seven days post-treatment.

Simple, inexpensive media for mass production of three entomophthoralean fungi.

The entomophthoralean fungi Batkoa sp., Furia sp. and Neozygites floridana have been suggested for biocontrol of insect pests: the first two for control of spittlebug pests of pasture and sugarcane, and the third for mites of agricultural importance. To develop these agents as biopesticides and bioacaricides, it is important to have available culture media that maximize production at low cost. The research reported here evaluates, in different combinations and concentrations, the effect of four complex sources of nitrogen on production of mycelium or hyphal bodies in liquid media of all three species. Yeast extract allowed the highest production of Batkoa sp., with a concentration of 0.5% being the most suitable for vegetative (mycelial) growth. The combination of 0.33% each of yeast extract + beef extract + skim milk allowed the highest production of Furia sp. Mycelium. The combination of yeast extract + skim milk (0.5% of each) allowed the second highest production of Furia sp., and was the most suitable for mass production due to the lower cost. The combination of 1 %each of yeast extract + peptone + skim milk was the most suitable for production of N. floridana hyphal bodies.

Effect of salts, vitamins, sugars and nitrogen sources on the growth of three genera of Entomophthorales: Batkoa, Furia, and Neozygites.

Entomophthorales pathogenic to insects and mites often cause epizootics in their host populations, but some have been difficult to culture in vitro and, therefore, to develop as biopesticides. Grace's insect cell culture medium supplemented with lactalbumin hydrolysate and yeastolate has allowed growth of several species which until recently were referred to as obligate parasites. The research reported here was designed to evaluate the effects of the salts, vitamins and amino acids used to prepare the insect cell culture medium on in vitro growth of Batkoa sp. and Furia sp., pathogens of the spittlebug pests of pasture and sugar-cane in Brazil, and Neozygites floridana, a pathogen of several mite species. Also, several sources of carbon and nitrogen were examined. Batkoa sp., Furia sp. and N. floridana were similar concerning their growth patterns in a basic medium with added salts, vitamins and amino acids, as well as with a combination of all three compoments. The addition of salts to the basic medium of sugars plus lactalbumen hydrolysate and yeastolate caused a significant increase in biomass production of the three fungal species. The addition of vitamins and amino acids had less effect. Batkoa sp., Furia sp. and N. floridana are similar in growth patterns in media with various sources of carbon, but different in media with different sources of nitrogen. The production of the three fungal species is significantly higher in medium containing 2.66% glucose than in medium with 2.66% sucrose. The addition of 0.1% monossacarides to media containing 2.66% sucrose did not significantly increase biomass production.