Efficacy of Purpureocillium lilacinum AUMC 10149 as biocontrol agent against root-knot nematode Meloidogyne incognita infecting tomato plant

Abstract Root-knot nematode Meloidogyne incognita is among the biotic factors which has greatly affected both the yield and the quality of the tomato crop. The egg parasitic nematode, Purpureocillium lilacinum (Pl) is considered as one of the most promising agents in controlling and overcoming this plant pathogen. The nematicidal effect of the native isolate Pl AUMC 10149 on second stage juveniles survival and egg hatching of M. incognita at different times of exposure was tested in vitro. The obtained data showed that Pl gave a maximum percentage of J2 mortality (97.6%) and egg hatching inhibition (79.8%) after 72 hours of exposure. The potentiality of Pl as well as Bio-Nematon to control M. incognita infecting tomato was conducted using different times of application in vivo. Nine treatments with five replicates were used for such bioagents compared with the nematicide Oxamyl. Each seedling was inoculated with 1000 J2s of nematode/pot and 10 mL of Pl (1x1010 CFU/mL) or Bio-Nematon spore suspension (1x108 CFU/mL) 10mL/pot. The results indicated that the most effective treatments in reducing nematode population, number of galls and egg masses of M. incognita in plant roots was performed with treatment by Pl pre-planting and post-infection with Pl (Rf 1.9) giving a significant enhancement in plant length (64.9%), fresh weight (72.52%) and shoot dry weight (163.41%) without negatively impacting environment. Therefore, the present study confirmed that using P. lilacinum AUMC 10149 can be used as a practical supplement to environmentally friendly disease management of root-knot nematodes in Egypt.

Resumo O nematoide-das-galhas Meloidogyne incognita está entre os fatores bióticos que afetaram enormemente a produção e a qualidade da cultura do tomate. O nematoide parasita de ovos, Purpureocillium lilacinum (Pl), é considerado um dos mais promissores agentes no controle e superação desse fitopatógeno. O efeito nematicida do isolado nativo Pl AUMC 10149 na sobrevivência de juvenis de segundo estágio e na eclosão dos ovos de M. incognita em diferentes momentos de exposição foi testado in vitro. Os dados obtidos mostraram que o Pl deu um percentual máximo de mortalidade de J2 (97.6%) e inibição da eclosão dos ovos (79.8%) após 72 horas de exposição. A potencialidade de Pl e de Bio-Nematon para controlar M. incognita infectando tomate foi conduzida em diferentes tempos de aplicação in vivo. Nove tratamentos com cinco repetições foram usados para tais bioagentes em comparação com o nematicida Oxamyl. Cada muda foi inoculada com 1.000 J2s de nematoide / vaso e 10 mL de Pl (1×1010 CFU/mL). Ou suspensão de esporos Bio-Nematon (1×108 CFU/mL) 10mL/pot. Os resultados indicaram que os tratamentos mais eficazes na redução da população de nematoides, número de galhas e desovas de M. incognita nas raízes das plantas foram realizados com Pl pré-plantio e pós-infecção com Pl (Rf 1.9), dando um aumento significativo no comprimento da planta (64.9%), massa fresca (72.52%) e massa seca da parte aérea (163.41%) sem impactar negativamente o meio ambiente. Portanto, o presente estudo confirmou que o uso de P. lilacinum AUMC 10149 pode ser usado como um suplemento prático para o manejo ecologicamente correto de nematoides-das-galhas no Egito.

Efficacy of Purpureocillium lilacinum AUMC 10149 as biocontrol agent against root-knot nematode Meloidogyne incognita infecting tomato plant / Eficácia de Purpureocillium lilacinum AUMC 10149 como agente de biocontrole contra o nematoide Meloidogyne incógnita que infecta plantas de tomate

Abstract Root-knot nematode Meloidogyne incognita is among the biotic factors which has greatly affected both the yield and the quality of the tomato crop. The egg parasitic nematode, Purpureocillium lilacinum (Pl) is considered as one of the most promising agents in controlling and overcoming this plant pathogen. The nematicidal effect of the native isolate Pl AUMC 10149 on second stage juvenile's survival and egg hatching of M. incognita at different times of exposure was tested in vitro. The obtained data showed that Pl gave a maximum percentage of J2 mortality (97.6%) and egg hatching inhibition (79.8%) after 72 hours of exposure. The potentiality of Pl as well as Bio-Nematon to control M. incognita infecting tomato was conducted using different times of application in vivo. Nine treatments with five replicates were used for such bioagents compared with the nematicide Oxamyl. Each seedling was inoculated with 1000 J2s of nematode/pot and 10 mL of Pl (1x1010 CFU/mL) or Bio-Nematon spore suspension (1x108 CFU/mL) 10mL/pot. The results indicated that the most effective treatments in reducing nematode population, number of galls and egg masses of M. incognita in plant roots was performed with treatment by Pl pre-planting and post-infection with Pl (Rf 1.9) giving a significant enhancement in plant length (64.9%), fresh weight (72.52%) and shoot dry weight (163.41%) without negatively impacting environment. Therefore, the present study confirmed that using P. lilacinum AUMC 10149 can be used as a practical supplement to environmentally friendly disease management of root-knot nematodes in Egypt.

Resumo O nematoide-das-galhas Meloidogyne incognita está entre os fatores bióticos que afetaram enormemente a produção e a qualidade da cultura do tomate. O nematoide parasita de ovos, Purpureocillium lilacinum (Pl), é considerado um dos mais promissores agentes no controle e superação desse fitopatógeno. O efeito nematicida do isolado nativo Pl AUMC 10149 na sobrevivência de juvenis de segundo estágio e na eclosão dos ovos de M. incognita em diferentes momentos de exposição foi testado in vitro. Os dados obtidos mostraram que o Pl deu um percentual máximo de mortalidade de J2 (97.6%) e inibição da eclosão dos ovos (79.8%) após 72 horas de exposição. A potencialidade de Pl e de Bio-Nematon para controlar M. incognita infectando tomate foi conduzida em diferentes tempos de aplicação in vivo. Nove tratamentos com cinco repetições foram usados ​​para tais bioagentes em comparação com o nematicida Oxamyl. Cada muda foi inoculada com 1.000 J2s de nematoide / vaso e 10 mL de Pl (1×1010 CFU/mL). Ou suspensão de esporos Bio-Nematon (1×108 CFU/mL) 10mL/pot. Os resultados indicaram que os tratamentos mais eficazes na redução da população de nematoides, número de galhas e desovas de M. incognita nas raízes das plantas foram realizados com Pl pré-plantio e pós-infecção com Pl (Rf 1.9), dando um aumento significativo no comprimento da planta (64.9%), massa fresca (72.52%) e massa seca da parte aérea (163.41%) sem impactar negativamente o meio ambiente. Portanto, o presente estudo confirmou que o uso de P. lilacinum AUMC 10149 pode ser usado como um suplemento prático para o manejo ecologicamente correto de nematoides-das-galhas no Egito.

Keratomycosis due to Purpureocillium lilacinum: A case report from Sub-Himalayan region of Uttarakhand

The fungus Purpureocillium lilacinum previously known as Paceliomyces lilacinus is an emerging pathogen that can cause severe human infections including devastating oculomycosis. Treatment with traditional antifungals often fails, and the organism shows variable susceptibility to novel triazoles. We hereby report a case of keratomycosis caused by Pur. lilacinum in an immunocompetent male patient following trauma. The patient was successfully treated with voriconazole. The drug shows good activity against Pur. lilacinum and could be a promising therapeutic alternative to treat infections caused by this fungus, which generally shows resistance to conventional antifungal agents including novel triazoles.

Endophytic fungus Purpureocillium sp. A5 protect mangrove plant Kandelia candel under copper stress

Abstract Mangrove is an important ecosystem in the world. Mangrove ecosystems have a large capacity in retaining heavy metals, and now they are usually considered as sinks for heavy metals. However, the mechanism of why the soil of mangrove ecosystems can retain heavy metal is not certain. In this research, endophytic fungus Purpureocillium sp. A5 was isolated and identified from the roots of Kandelia candel. When this fungus was added, it protected the growth of K. candel under Cu stress. This can be illustrated by analyzing chlorophyll A and B, RWC and WSD to leaves of K. candel. Purpureocillium sp. A5 reduces uptake of Cu in K. candel and changes the pH characterization of soil. Furthermore, A5 increase the concentration of Cu complexes in soil, and it enhanced the concentration of carbonate-bound Cu, Mn-Fe complexes Cu and organic-bound Cu in soil. Nevertheless, a significant reduction of the Cu ion was noted among A5-treated plants. This study is significant and illustrates a promising potential use for environmental remediation of endophytes, and also may partially explain the large capacity of mangrove ecosystems in retaining heavy metals.

A Case of Localized Skin Infection due to Purpureocillium lilacinum / 대한의진균학회지

Purpureocillium lilacinum, formerly Paecilomyces lilacinus, is a saprophytic fungus found in soil and rotting vegetation and rarely pathogenic to humans. Only 4 cases of cutaneous infection caused by Purpureocillium lilacinum have been reported in the name of Paecilomyces lilacinus. Herein, we report a case of localized cutaneous infection due to Purpureocillium lilacinum. A healthy 81-year-old immunocompetent male presented with an erythematous scaly and pustular plaque on his left dorsal hand that had begun 3 months ago. Histopathologic examination showed suppurative granulomatous inflammation with hyphae and round spores in the dermis. Periodic acid-Schiff and methenamine silver stain revealed fungal spores. Fungus culture from the biopsy specimen revealed velvety pink to white colonies after 15 day-incubation period. The slide culture stained with lactophenol-cotton blue showed typical long hyphae and flask-shaped phialides with oval conidia in chains. The result of DNA sequencing from the colony was identical to that of Purpureocillium lilacinum.

A Case of Fungal Keratitis Caused by Purpureocillium lilacinum: A Microbiological Review of Korean Cases / 대한의진균학회지

Purpureocillium(P) lilacinum is a ubiquitous, saprophytic filamentous fungus that is infrequently reported in keratitis and cutaneous infections. However, the microbiological characterization of the culture isolates is limited in Korea. A 56-year-old male who suffered a pine needlestick to his right eye 10 days previously presented with ocular opacity and pain. A microscopic examination of a corneal scraping by Gram staining and calcofluor white staining was negative for bacteria and fungi. Fungal culture yielded pure white cottony molds on Sabouraud's dextrose agar after a 3-day incubation. Microscopic examination further revealed a mixture of a verticillate arrangement of phialides resembling the Penicillium structure and sparsely branched conidiophores bearing single to small clusters of conidia. This was initially presumed to be a species of Penicillium but the colonies never turned green with further incubation. It was subsequently identified as P. lilacinum by 28S rDNA sequencing and MALDI-TOF mass spectrometry. Antifungal susceptibility test revealed that this organism was resistant to flucytosine, amphotericin B, fluconazole, itraconazole, voriconazole, and caspofungin. After treatment with topical 5% voriconazole and oral itrazonazole combined with multiple debridements for 2 weeks, the patient was discharged with improved visual acuity. We thus report the first case of P. lilacinum infection that required molecular identification due to mixed conidiogenesis features and that showed laboratory-confirmed antifungal resistance in Korea.

Ocurrencia natural de hongos entomopatógenos en suelos cultivados con yerba mate (Ilex paraguariensis St. Hil.) en Misiones, Argentina / Natural ocurrence of entomopathogenic fungi in soils cultivated with Paraguay tea (Ilex paraguariensis St. Hil.) in Misiones, Argentina

Este trabajo tuvo por objetivo aislar, identificar y caracterizar morfológicamente hongos entomopatógenos presentes en suelos cultivados con yerba mate (Ilex paraguariensis St. Hil.). Se realizó una prospección de hongos entomopatógenos nativos a partir de 40 muestras de suelos cultivados con yerba mate ubicados en la provincia de Misiones, República Argentina, desde mayo de 2008 hasta junio de 2010. Las muestras de suelo se obtuvieron en cercanías de plantas de yerba mate, se aplicó la técnica de dilución seriada en medios de cultivos selectivos. Después de obtenidos los cultivos puros, los hongos fueron caracterizados macroscópica y microscópicamente, y se los identificó mediante el uso de claves taxonómicas específicas. Se aislaron 29 cepas fúngicas pertenecientes a las especies Beauveria bassiana (n = 17), Metarhizium anisopliae (n = 2) y Purpureocillium lilacinum (n = 10)

This study aimed to morphologically isolate, identify and characterize entomopathogenic fungi present in soils cultivated with Paraguay tea (Ilex paraguariensis). A survey of native entomopathogenic fungi was conducted from 40 soil samples grown with Paraguay tea in the province of Misiones, Argentina, from May 2008 to June 2010. The soil dilution plate methodology on selective culture media was used to isolate microorganisms. Taxonomic identification was performed using macroscopic and microscopic characters and specific keys. Twenty nine strains, belonging to the species Beauveria bassiana (n = 17), Metarhizium anisopliae (n = 2) and Purpureocillium lilacinum (n = 10) were isolated and identified

Efecto del filtrado crudo de Purpureocillium sp. (Cepa UdeA0106), sobre la eclosión de huevos y movilidad de juveniles de Meloidogyne incognita-javanica / Effect of the raw filtrate of Purpureocillium sp. (UdeA0106 strain) on the eclosion of eggs and the motility of Meloidogyne incognitajavanica juveniles

Meloidogyne incognita-javanica representa una limitación de los cultivos agrícolas y es necesario buscar alternativas para su manejo. El presente trabajo tuvo como objetivo evaluar el efecto in vitro del filtrado crudo de Purpureocillium sp. (Cepa UdeA0106) sobre laeclosión de huevos y los estadios juveniles de Meloidogyne spp. Adicionalmente, se evaluaron los medios: caldo extracto de malta (CEM), caldo extracto de levadura (CEL) y caldo papa glucosa (CPG), escogiéndose aquel que no ejerciera daño sobre la morfología de los huevos y permitiera la eclosión de los estadios juveniles. El hongo se multiplicó durante 7 días y fue filtrado. Para establecer su efecto sobre los huevos y estadios J2 de Meloidogyne spp., se probaron concentraciones del filtrado al 100 %, 90 %, 70 %, 50 %, 25 % y 10 %; comparándolas con un control compuesto de estadios J2 en agua destilada estéril (ADE) y CPG. Las variables a evaluar fueron el porcentaje de eclosión e inmovilidad en los J2. La unidad experimental fue la caja de Petri, cada una con 200 huevos/J2, con 5 réplicas de cada tratamiento. Se utilizó un diseño experimental completamente aleatorizado con ANOVA (P = 0,05) y análisis de comparación múltiple de Tukey. Se encontraron diferencias significativas entre todos los tratamientos (P < 0,05) pero el tratamiento correspondiente al filtrado puro (100 %) logró mantener el porcentaje más bajo de eclosión y mayor porcentaje de inmovilidad de los J2 (96,8 %). Las concentraciones al 90 % y 70 % igualmente lograron efectos en los J2, con porcentajes altos de inmovilidad.

Meloidogyne incognita-javanica represents an important threat to the agricultural crops, and it is necessary to seek alternatives for its control. The aim of this research it was to evaluate a crude filtrate from the strain Purpureocillium sp. UdeA0106 in order to establish the in vitro effect on the egg hatching and the immobility of juvenile stages of Meloidogyne spp. The mediums evaluated were malt extract broth (MEB), yeast extract broth (YEB), and potato dextrose broth (PDB), after which it was chosen the one that didn't produce any damages on nematode eggs and allowed the emergence of the larval stages. The fungus was multiplied for 7 days, and then it was filtrated. To evaluate its effect on the eggs and the J2 of Meloidogyne spp., filtrate concentrations were made at 100 %, 90 %, 70 %, 50 %, 25 % and 10 %, contrasting them with a control composed of the J2 stages in sterile distilled water and PDB. The variables evaluated were the percentage of altered eggs and the immobility observed in J2 stages. The experimental unit was the Petri dish, each one containing 200 eggs /J2. Each treatment was replicated 5 times. It was used a completely randomized design, and an ANOVA was performed (P = 0,05) as well as a multiple comparison test (Tukey). Significant differences were found between all treatments (P < 0,05), although the most effective in inhibiting both egg hatching and J2 mobility (96,8 %) were the full strength filtering (100 %). The dilutions at 90 % and 70 %, were able to produce effects with high percentages in the immobility.

MANEJO INTEGRADO DE NEMATODOS FITOPARÁSITOS EN ALMÁCIGOS DE PLÁTANO DOMINICO HARTÓN (Musa AAB SIMMONDS) / INTEGRATED MANAGEMENT OF PHYTOPARASITIC NEMATODES IN DOMINICO HARTÓN PLANTAIN (Musa AAB SIMMONDS) SEEDLINGS

Como consecuencia de la alimentación de Radopholus similis, se generan daños en las raíces y el cormo que conllevan a un crecimiento deficiente de las plantas y reducción en rendimiento hasta 80%. Por tal motivo, el objetivo de esta investigación fue determinar el efecto de la integración de prácticas de manejo como limpieza sanitaria de cormos y biológicas como Micorrizas Arbusculares (MA), Purpureocillium lilacinum y Bacillus subtilis sobre el crecimiento de las plántulas de Dominico Hartón y la población de nematodos fitoparásitos. El experimento se realizó en la granja Montelindo de la Universidad de Caldas, a 1010 msnm, 23ºC de temperatura promedio y 1800 mm de precipitación. Los tratamientos fueron asignados en un diseño experimental completamente aleatorio, cada uno conformado por 20 cormos (unidades experimentales) sembrados en bolsas negras de 5 kg de capacidad, así: cormos con limpieza sanitaria sin aplicación de productos (T1), cormos con limpieza sanitaria + MA (T2), cormos con limpieza sanitaria + P. lilacinum (T3), cormos con limpieza sanitaria + B. subtilis (T4), y cormos con limpieza sanitaria + MA + P. lilacinum + B. subtilis (T5). El anterior procedimiento también se realizó a cormos tradicionales sembrados en suelo sin esterilizar (T6 a T10, respectivamente). En los tratamientos que fueron establecidos en suelo esterilizado y cormos con limpieza sanitaria, no hubo presencia de individuos de los principales géneros de nematodos fitoparásitos del plátano como Radopholus, Meloidogyne y Helicotylenchus. Los tratamientos en los cuales se utilizó suelo infestado y cormos con limpieza sanitaria, presentaron una disminución de hasta un 64% en la población de nematodos fitoparásitos en comparación con la inicial, aunque mostraron también una cantidad de nematodos menor al testigo. De estos tratamientos el que menor población presentó fue el T10 (Suelo infestado + cormos con limpieza sanitaria + MA + P. lilacinum + B. subtilis) con una población promedio de 472 nematodos fitoparásitos en 100 g de suelo. La integración de todas las medidas de manejo resultó más eficiente en el control de los nematodos fitoparásitos en plátano, con o sin la esterilización del suelo; y la limpieza sanitaria de cormos no afectó negativamente el desarrollo de las raíces ni de la parte aérea de las plantas, pues no se presentaron diferencias significativas respecto a los tratamientos donde se usaron cormos tradicionales.

As a result of feeding of Radopholus similis, both roots and corms are damaged and this results in plant growth deficiency and yield losses up to 80%. Therefore, the objective of this study was to determine the effect of the integration of management practices such as corms sanitary cleaning and biological cleaning such as Arbuscular mycorrhizae (AM), Purpureocillium lilacinum and Bacillus subtilis on the development of Dominico Hartón plantain seedlings and phytoparasitic nematods populations. The experiment was conducted at the Universidad de Caldas Montelindo farm located at 1,010 m.a.s.l.., average temperature 23ºC and 1,800 mm rainfall. The treatments were assigned in a completely randomized block design, each one with 20 corms (experimental units) planted in 5 kg black plastic bags as follows: corms with sanitary seed cleaning without application of products (T1); corms with sanitary cleaning + AM (T2); corms with sanitary cleaning + P. lilacinum (T3); corms with sanitary cleaning + B. subtilis (T4); and corms with sanitary cleaning + AM + P. lilacinum + B. subtilis (T5). The described procedure was also applied to traditional corms planted in soil without sterilization (T6 to T10, respectively). In the treatments established in sterilized soil and cleaned corms, there was not presence of individuals of the main plant parasitic nematodes of plantain, such as, Radopholus, Meloidogyne and Helicotylenchus. Treatments with infested soil with nematodes and cleaned corms, showed a reduction in plant parasitic nematodes up to 64% compared with the initial population, although they also showed lower amount of nematodes than the control population. From these treatments, the one that had the lowest population of nematodes was the T10 (Infested soil + cleaned corms + AM + P. lilacinum + B. subtilis) with an average population of 472 phytoparasitic nematodes/100 g of soil). The integration of all management practices resulted in the most efficient way to control the plant phytoparasitic nematodes of plantain, with or without soil sterilization, and the corms sanitary cleanness did not affect either the root development or the aerial parts of the seedlings, since there were not significant differences with respect to the treatments using traditional corms.

Interaction of an opportunistic fungus Purpureocillium lilacinum with human macrophages and dendritic cells

Introduction Purpureocillium lilacinum is emerging as a causal agent of hyalohyphomycosis that is refractory to antifungal drugs; however, the pathogenic mechanisms underlying P. lilacinum infection are not understood. In this study, we investigated the interaction of P. lilacinum conidia with human macrophages and dendritic cells in vitro. Methods Spores of a P. lilacinum clinical isolate were obtained by chill-heat shock. Mononuclear cells were isolated from eight healthy individuals. Monocytes were separated by cold aggregation and differentiated into macrophages by incubation for 7 to 10 days at 37°C or into dendritic cells by the addition of the cytokines human granulocyte-macrophage colony stimulating factor and interleukin-4. Conidial suspension was added to the human cells at 1:1, 2:1, and 5:1 (conidia:cells) ratios for 1h, 6h, and 24h, and the infection was evaluated by Giemsa staining and light microscopy. Results After 1h interaction, P. lilacinum conidia were internalized by human cells and after 6h contact, some conidia became inflated. After 24h interaction, the conidia produced germ tubes and hyphae, leading to the disruption of macrophage and dendritic cell membranes. The infection rate analyzed after 6h incubation of P. lilacinum conidia with cells at 2:1 and 1:1 ratios was 76.5% and 25.5%, respectively, for macrophages and 54.3% and 19.5%, respectively, for cultured dendritic cells. Conclusions P. lilacinum conidia are capable of infecting and destroying both macrophages and dendritic cells, clearly demonstrating the ability of this pathogenic fungus to invade human phagocytic cells. .

First Report on Isolation of Penicillium adametzioides and Purpureocillium lilacinum from Decayed Fruit of Cheongsoo Grapes in Korea

Two species, Penicillium adametzioides and Purpureocillium lilacinum, were isolated from decayed grapes (cv. Cheongsoo) in Korea. Each species was initially identified by phylogenetic analysis of a combined dataset of two genes. Internal transcribed spacer (ITS) and beta-tubulin (BT2) genes were used for identification of Penicillium adametzioides, and ITS and partial translation elongation factor 1-alpha (TEF) genes were used for identification of Purpureocillium lilacinum. Morphologically, they were found to be identical to previous descriptions. The two species presented here have not been previously reported in Korea.