Medicinal Plant-Derived Copper Nanoparticles Effectively Manage Early Blight Disease in Tomato: An In vitro Study

Aims: Early blight of tomato (Solanum lycopersicum L.) incited by Alternaria solani is an economically important disease. This paper analyses the efficacy of copper nanoparticles (Cu-NPs) as an alternative to its existing management practices that predominantly rely on toxic fungicides which are potentially harmful to humans and the environment.Study Design: The experiment was conducted in a completely randomized block design with 7 treatments viz. T1:Cu-NPs from tulsi leaves, T2:Cu-NPs from neem leaves, T3:Cu-NPs from bael leaves, T4: Cu-NPs from chemical A (prepared from 0.2 M of Cu(NO3)2.3H2O + 0.2 M Ascorbic acid), T5: Cu-NPs from chemical B (0.2 M Cu(NO3)2.3H2O + 0.2 M CuCl2 + 0.4 N NH4OH), T6: Carbendazim 50 WP as check and T7: Control. Each treatment was tested at two levels of in vitro concentrations (i.e. 300 and 500 ppm) and the treatments were replicated thrice.Place and Duration of Study: Considering the importance of the disease and for developing an effective management strategy, the present study was carried out in the Department of Plant Pathology, College of Agriculture, Odisha University of Agriculture and Technology, Bhubaneswar during 2020-21.Methodology: Stable Cu-NPs were synthesized through three green synthesis methods by using bael (Agel marmelos L.), tulsi (Ocimum sanctum L.), and neem (Azadirachta indica L.) leaves and two chemical synthesis methods (i.e. chemical A and B).Results: The size (diameter) of the Cu-NPs from Agel marmelos, Ocimum sanctum, and Azadirachta indica were 427.4, 1,019, and 246.5 nm, and that from the aqueous solution reduction method and precipitation method were 1,537 and 867.8 nm, respectively. The synthesized Cu-NPs were evaluated against Alternaria solani at 300 and 500 ppm while Carbendazim 50 WP served as the standard check. The Azadirachta indica leaf-derived Cu-NPs at 300 and 500 ppm in the in vitro potato dextrose ager (PDA) medium exhibited the maximum mycelial growth inhibition of 60.24% and 75.9%, respectively. The size of Cu-NPs showed an inverse relation with the antifungal efficacy that indicated greater cellular penetration of smaller nanoparticles whereas much lower inhibition of mycelial growth in Carbendazim 50 WP as a check showed the lowest growth inhibition of 39.75% (300 ppm) and 59.03% (500 ppm).Conclusion: The results thus indicated higher efficacy of nanoformulations at lower concentrations that can be effectively used for the development of new nano-based plant protection agents against the early blight of tomatoes.

Effect of pH on the Mycelial Growth of Aspergillus niger and Aspergillus flavus

Aspergillus fungus is well-known for causing a number of secondary plant and food rots that can result in the buildup of mycotoxins, despite their significant economic contribution to the fermentation industries. In the past, even the most fundamental influences on the development and inhibition of significant mycotoxigenic fungi were only partially understood. To overcome this issue, an investigation on the impact of pH on the mycelial development of Aspergillus species was conducted. A. niger and A. flavus were inoculated in PDA medium with a range of pH values from 4.0 to 9.0 under in vitro conditions and then cultured for 7 days at room temperature. Results revealed that, the mycelial growth of A. niger and A. flavus was increased with increase in pH level up to 6 and then became static up to pH level 9 and there was no mycelial growth in pH 4. Hence, it was concluded that the use of alkaline compounds as a way of preventing their proliferation and reproductive processes may be used to inhibit the development and spore production of A. niger and A. flavus, which can affect the crops.

Evaluación del cultivo in vitro de una cepa mexicana de Ganoderma lucidum / Evaluation of in vitro cultivation of a Mexican strain of Ganoderma lucidum

RESUMEN La cepa mexicana CP-145 de Ganoderma lucidum debido a la importancia medicinal que ha presentado últimamente, la presente investigación tuvo como objetivo evaluar el efecto de la temperatura y medio de cultivo sobre el crecimiento micelial óptimo en diferentes rangos de pH. Los tratamientos correspondieron en la utilización del medio de cultivo papa dextrosa agar (PDA) y extracto de malta agar (EMA), con dos niveles de temperatura (25 y 28 °C) y seis rangos de pH (4.0, 4.5, 5.0, 5.5, 6.0 y 6.5). El diseño experimental fue completamente al azar con medidas repetidas a través del tiempo, analizados con el paquete REPEATED MEASURE y el efecto tiempo con PROC MIXED de SAS. Como resultado se obtuvieron que el efecto de la temperatura y medios de cultivo en los diferentes rangos de pH, presentaron diferencias significativas (P ≤ 0.05). El crecimiento micelial óptimo de la cepa mexicana de G. lucidum fue en el medio de cultivo EMA en los rangos de pH de 4.0 y 4.5 con 8.3 y 8.2 cm respectivamente. De igual forma, en los rangos de pH 4.0 y 4.5 se obtuvieron los crecimientos miceliales óptimos a temperatura de 25 °C con 8.1 y 8.0 cm respectivamente. El cual concluyó esta investigación que el crecimiento micelial óptimo de la cepa mexicana fueron a pH 4.0 y 4.5, temperatura de 25 °C y medio de cultivo EMA.

ABSTRACT The Mexican strain CP-145 of Ganoderma lucidum due to the medicinal importance it has presented lately, the present investigation had as objective to evaluate the effect of temperature and culture medium on the optimal mycelial growth in different pH ranges. The treatments corresponded to the use of potato dextrose agar (PDA) and malt extract agar (EMA), with two temperature levels (25 and 28 °C) and six pH ranges (4.0, 4.5, 5.0, 5.5, 5.5, 6.0 and 6.5). The experimental design was completely randomised with repeated measures over time, analysed with the REPEATED MEASURE package and the time effect with PROC MIXED of SAS. As a result, the effect of temperature and culture media in the different pH ranges showed significant differences (P ≤ 0.05). The optimal mycelial growth of the Mexican strain of G. lucidum was in the EMA culture medium in the pH ranges of 4.0 and 4.5 with 8.3 and 8.2 cm respectively. Similarly, in the pH ranges 4.0 and 4.5 the optimum mycelial growth was obtained at 25 °C with 8.1 and 8.0 cm respectively. This research concluded that the optimal mycelial growth of the Mexican strain was at pH 4.0 and 4.5, temperature of 25 °C and EMA culture medium.

Sewage Sludge Biochar Effects on Phytopathogenic Fungi and Beneficial Microorganisms

Abstract Biochar (BCH) is a solid product, rich in carbon, obtained by heating biomass under controlled conditions of oxygenation, in a process known as pyrolysis. Its benefits are associated with improvements to the physical, chemical and biological properties of soil. Furthermore, BCH can affect the growth of phytopathogenic microorganisms. Despite research advances in this area, there is still a lack of information on the effect of BCH concentration on different soilborne microorganisms. This work evaluated the direct effect of sewage sludge biochar (SSB) on the in vitro growth of different soilborne fungi. Eight phytopathogens [Fusarium oxysporum, F. oxysporum f. sp. lycopersici (CEN 1456), Macrophomina phaseolina (429), Sclerotinia sclerotiorum, S. sclerotiorum (CEN 1147), Sclerotium rolfsii (CEN 216), Sclerotium cepivorum (CEN 1357), Rhizoctonia solani] were evaluated. Additionally, a biological control agent [Trichoderma afroharzianum (T-22)] was also studied. Microorganisms were subjected to growth in PDA (Potato-Dextrose-Agar) culture medium enriched with SSB. Biochars, produced at 300 and 500 ºC, were applied at different doses: 0.0 (control), 0.2, 0.4, 0.6, 0.8 and 1.0 (g of SSB/100 ml of PDA). Biochars showed inhibitory effects on the mycelial growth of the different fungi evaluated. It was observed that there is a certain specificity of biochar concentration that must be evaluated for the control of phytopathogens. In most cases, the 0.4 and 0.6% concentrations had a greater inhibitory effect on phytopathogens and did not affect the biological control agent. Therefore, SSB proved to be a promising product for the control of different soilborne phytopathogens.

Evaluation of hybrids and single spore isolates of paddy straw mushroom Volvariella volvacea (Bull.) Singer for fruiting body yield and nutritional quality

Aim: Developing high yielding single spore isolates and hybrid strains of paddy straw mushroom (Volvariella volvacea) with superior nutritional composition of the fruiting bodies.Methodology: Two contrasting high yielding strains (DMRO-463 and DMRO-484) of V. volvacea previously released for higher fruiting body yield were used for isolation of single spore isolates (SSIs). The slow growing SSIs were used for developing the hybrid strains by mating on Malt Extract Agar Medium Petri dishes. Conversely, the fast growing SSIs and the developed hybrid strains were screened for downward mycelial growth on paddy straw filled in wide mouth test tubes. Based upon downward mycelial growth, only eight SSIs and ten hybrid strains with numerically higher downward mycelial growth compared to two parents' checks were further evaluated for fruiting body yield on composted cotton ginning mill waste substrate under indoor conditions. Results: In successive yield evaluation trials (one preliminary and three full scale), hybrid strains VvH-11, VvH-13 and VvH-18 gave fruiting body yield higher than the two parents. Out of these hybrids, the yield distribution at different height (selves) in growing room was consistent in hybrid VvH-13 in trial-2, while rest two hybrids and parents showed significantly lower yield in lowest self (30 cm above floor level). The fruiting body weight also showed similar trend. The fruiting bodies from the highest yielding hybrid VvH-11 exhibited highest level of crude fiber (2.07%) and ash (10.95%) contents, while the hybrid VvH-13 giving consistent yield across growing room exhibited higher level of crude fiber (2.00 %), vitamin C (52.35 mg 100 g-1) and vitamin D (1434.7 µg 100 g-1). The third hybrid VvH-18 was superior in crude fiber (1.74%), manganese (22.46 ppm), selenium (0.26 ppm) and vitamin C (43.13 mg 100 g-1). All three hybrids exhibited lower fat (1.54 to 1.86 %) content compared to parents (2.54 to 2.59 %). Interpretation: High yielding hybrid strains can be developed in paddy straw mushroom (V. volvacea) through mating of slow growing SSIs, and their screening involving downward mycelial growth on paddy straw and repetitive grow out trials.

Obtaining monokaryotic and dikaryotic mycelial cultures of two Amazonian strains of Geastrum (Geastraceae, Basidiomycota)

The high diversity of the genus Geastrum and the difficulty of obtaining mycelial cultures impairs the study of the ecophysiology and the exploration of the biotechnological potential of the taxon. In this study, different culture media were tested to obtain mycelial cultures for G. lloydianum and G. subiculosum collected in the Brazilian Amazon. Data on spore germination, and isolation of monokaryotic cultures and in vitro sexual reproduction are presented, as well as a brief morphological description of the cultures obtained. For both species, Potato Dextrose Agar (PDA) was the most promising of the tested culture media. The highest growth in agar culture ever recorded for this genus is reported (4.9 mm per week for G. lloydianum and 7.5 mm for G. subiculosum). In the PDA culture medium, spores germinated after 35-40 days of incubation and the isolation of monokaryotic cultures of the two species, as well as in vitro sexual crosses, were successfully performed. (AU)

Antifungal potential of crude extracts of Trichoderma spp / Potencial antifúngico de extratos brutos de Trichoderma spp

Abstract Antibiosis is the mechanism by which certain microorganisms respond to the presence of others, secreting compounds or metabolites capable of inhibiting or impeding their development. The crude extract of Trichoderma contains a mixture of secondary compounds, which may show antibiotic effect, and has been used for the prospect of this fungus for biological control and other industrial purposes. Faced with the increasing demand of agriculture for ecologically compatible alternatives for the management of diseases, this work aimed to investigate the spectrum of action of Non-Volatile Metabolites (NVMs) of Trichoderma isolates against different plant pathogenic fungi. The antagonistic potential of NVMs was evaluated through the incorporation method of the filtered liquid extract in PDA medium. The assays showed that all the NVMs produced inhibited the fungus Sclerotinia sclerotiorum similarly. On the other hand, strains CEN1245 and CEN1274, both belonging to the species Trichoderma brevicompactum, showed broad spectrum against Sclerotium rolfsii, Colletotrichum gloesporioides, Verticillium dahliae, Fusarium oxysporum and Cylindrocladium sp. The present study describes isolates producing non-volatile metabolites with broad spectrum of antifungal action, as well as pathogen-specific. The Trichoderma spp. NVMs obtained from different soil samples cultivated with vegetables, cassava and maize were efficient in inhibiting plant pathogenic fungi belonging to other patossystems, such as forest or fruit, which could increase their potential application in biological control of plant diseases. In addition, these antagonistic fungi should be studied in greater depth for the identification of bioactive molecules of industrial interest or in commercial formulations of products for biological control of plant pathogens.

Resumo Antibiose é um mecanismo pelo qual certos microrganismos respondem à presença de outros, secretando compostos ou metabólitos capazes de inibir ou impedir o seu desenvolvimento. O extrato bruto de Trichoderma contém uma mistura de compostos secundários e tem sido utilizado na prospecção deste fungo para o controle biológico e demais fins industriais. Diante da crescente demanda da agricultura por alternativas ecologicamente compatíveis para o manejo de doenças, este trabalho teve como objetivo investigar o espectro de ação de Metabólitos Não Voláteis (MNVs), produzidos por isolados de Trichoderma, contra diferentes fungos fitopatogênicos. O potencial antagônico dos MNVs foi avaliado através do método de incorporação do extrato líquido filtrado em meio BDA. Os ensaios mostraram que todos os MNVs produzidos inibiram de forma semelhante o fungo Sclerotinia sclerotiorum. Por outro lado, os isolados CEN1245 e CEN1274, ambos Trichoderma brevicompactum, mostraram um amplo espectro de ação, atuando contra Sclerotium rolfsii, Colletotrichum gloesporioides, Verticillium dahliae, Fusarium oxysporum e Cylindrocladium sp. O presente estudo descreve isolados que produziram metabólitos não voláteis com amplo espectro de ação antifúngico, assim como patógeno-específico. Os MNVs de Trichoderma spp. obtidos de diferentes amostras de solo cultivadas com vegetais, mandioca e milho, foram eficientes na inibição de fungos fitopatogênicos pertencentes a outros patossistemas, como os de espécies florestais e frutíferas, o que poderia aumentar sua potencial aplicação no controle de doenças de plantas. Adicionalmente, estes fungos antagonistas devem ser mais bem estudados para identificação de moléculas bioativas de interesse industrial ou formulação de produtos para o controle biológico de patógenos de plantas.

Effect of surfactants and temperature on germination and vegetative growth of Beauveria bassiana

Three non-ionic surfactants: Tween20, Tween80 and Breakthru® were screened for their effects on spore germination and mycelial growth rates and for their influence on three isolates of Beauveria bassiana spore germination at various temperatures. Tween20 and Tween80 were compatible with all the B. bassiana isolates in the germination studies, but inhibited germination at higher surfactant concentrations, irrespective of the conidial concentrations. Breakthru® had an inhibitory effect on germination even at the lowest concentration of 0.1% on all the B. bassiana isolates. The effects of the surfactants on spore germination did not correspond with their effects on colony growth. Conidial viability within the same formulation declined significantly with increases in temperature, irrespective of the surfactant. The optimal temperature for conidial germination of B. bassiana isolates was approximately 25 °C with an upper limit at 30 °C. Isolate 7320 was identified as the least affected by the different surfactants. This isolate was able to germinate rapidly in a broad temperature range of 25–30 °C after 24 h, this characteristic being an essential factor in controlling house fly populations in poultry houses.

Morphology and mycelial growth rate of Pleurotus spp. strains from the Mexican mixtec region

Two native Pleurotus spp. strains (white LB-050 and pale pink LB-051) were isolated from rotten tree trunks of cazahuate (Ipomoea murucoides) from the Mexican Mixtec Region. Both strains were chemically dedikaryotized to obtain their symmetrical monokaryotic components (neohaplonts). This was achieved employing homogenization time periods from 60 to 65 s, and 3 day incubation at 28 °C in a peptone-glucose solution (PGS). Pairing of compatible neohaplonts resulted in 56 hybrid strains which were classified into the four following hybrid types: (R1-n xB1-n, R1-n xB2-1, R2-n xB1-n and R2-n xB2-1). The mycelial growth of Pleurotus spp. monokaryotic and dikaryotic strains showed differences in texture (cottony or floccose), growth (scarce, regular or abundant), density (high, regular or low), and pigmentation (off-white, white or pale pink). To determine the rate and the amount of mycelium growth in malt extract agar at 28 °C, the diameter of the colony was measured every 24 h until the Petri dish was completely colonized. A linear model had the best fit to the mycelial growth kinetics. A direct relationship between mycelial morphology and growth rate was observed. Cottony mycelium presented significantly higher growth rates (p < 0.01) in comparison with floccose mycelium. Thus, mycelial morphology can be used as criterion to select which pairs must be used for optimizing compatible-mating studies. Hybrids resulting from cottony neohaplonts maintained the characteristically high growth rates of their parental strains with the hybrid R1-n xB1-n being faster than the latter.

Efeito de fungicidas e herbicidas no controle de Sclerotinia sclerotiorum / Effect of fungicides and herbicides in the control of Sclerotinia sclerotiorum

O objetivo deste trabalho foi estudar o efeito de fungicidas e herbicidas sobre o crescimento micelial de S. sclerotiorum, e o efeito preventivo e curativo de fungicidas no controle da podridão branca da haste da soja. No experimento in vitro, vinte e oito fungicidas e quatro herbicidas, na concentração de 100 µg.i.a.mL-1, foram incorporados ao meio BDA e avaliados sobre o crescimento micelial de dois isolados. No experimento em campo, os fungicidas fluazinam, epoxiconazol+piraclostrobina, tebuconazol+trifloxistrobina, tebuconazol, ciproconazol+propiconazol, procimidona, iprodiona, vinclozolin, protioconazol, azoxistrobina+ciproconazol e tiofanato metílico, foram testados preventiva e curativamente no controle da doença. Com exceção de azoxistrobina, clorothalonil, pencycuron, quintozeno, tiofanato metílico, carboxin+thiram e carbendazim, todos os outros fungicidas inibiram entre 98% a 100% do crescimento micelial dos dois isolados. Dentre os herbicidas, clorimurom-etílico reduziu em 79,4% e 84,1% o crescimento micelial dos isolados. Os fungicidas protioconazol, iprodiona, ciproconazol+propiconazol, fluazinam, vinclozolin e epoxiconazol+piraclostrobina proporcionaram menor severidade da doença quando aplicados preventivamente. Em aplicações curativas, apenas iprodione destacou-se na redução da doença.

This study evaluated the effect of fungicides and herbicides on mycelial growth of S. sclerotiorum, and the preventive and curative effects of fungicides on the control of soybean stem white rot. Twenty eight fungicides and four herbicides were used in the in vitro experiment, at the concentration of 100 µg a.i. mL-1, incorporated into the medium PDA, and evaluated on the mycelial growth of two isolates. The fungicides fluazinam, epoxiconazole+piraclostrobine, tebuconazole+trifloxistrobine, tebuconazole, ciproconazole+propiconazole, procimidone, Biosci. J., Uberlândia, v. 29, n. 6 , p. 1989-1996, Nov./Dec. 2013 iprodione, vinclozolin, protioconazole, azoxistrobine+ciproconazole and methyl thiophanate were tested in the field for the preventive and curative control of the disease. Except for the fungicides azoxistrobine, clorothalonil, pencycuron, quintozene, methyl thiophanate, carboxin+thiram and carbendazim, all others inhibited from 98% to 100% of the mycelial growth of both isolates. Among the herbicides, clorimurom-ethyl reduced from 79.4% to 84.1% the growth of both isolates. The fungicides protioconazol, iprodione, ciproconazole+propiconazole, fluazinam, vinclozolin and epoxiconazole+piraclostrobine provided lower disease severity in prventive application; in contrast, only iprodione was effective in curative effect of disease reduction.

Efeito de óleos essenciais de plantas medicinais sobre a antracnose do sorgo / Effect of essential oils of medicinal plants on the anthracnose of sorghum

Objetivou-se avaliar a fungitoxicidade in vitro dos óleos essenciais de capim-limão, citronela, erva-cidreira e hortelã-pimenta sobre o fungo Colletotrichum graminicola (Ensaio I) e o efeito in vivo desses óleos e do óleo comercial de nim sobre a antracnose do sorgo (Ensaio II). No Ensaio I avaliou-se, em cinco épocas (dois, quatro, seis, oito e dez dias de incubação), o crescimento micelial do C. graminicola sob cinco concentrações dos óleos essenciais (C1= 0,25 µL mL-1; C2= 0,50 µL mL-1; C3= 0,75 µL mL-1; C4= 1,0 µL mL-1; C5= 1,25 µL mL-1). No Ensaio II, para avaliar o efeito preventivo, utilizou-se cinco tipos de óleos (capim-limão, citronela, erva-cidreira, hortelã-pimenta e nim) em quatro concentrações (2,5, 5,0, 7,5 e 10,0 µL mL-1). Para o efeito curativo, foram utilizados os mesmos óleos do efeito preventivo em duas concentrações (2,5 e 5,0 µL mL-1). Os óleos essenciais de C. nardus e C. citratus foram os mais eficientes na redução do crescimento micelial de C. graminicola (Ensaio I). No Ensaio II, no efeito preventivo, tanto os óleos essenciais quanto as concentrações reduziram significativamente a severidade da doença, destacando-se os óleos de capim-limão, erva-cidreira e nim. No efeito curativo, os tratamentos reduziram a severidade, porém não apresentaram diferença significativa entre si.

The objective was to evaluate the in vitro fungitoxicity of essential oils of lemongrass, citronella, lemongrass and peppermint on the fungus Colletotrichum graminicola (Test I) and in vivo effects of these oils and neem oil trade on the anthracnose sorghum (Test II). In Test I was evaluated in five seasons (two, four, six, eight and ten days of incubation), the mycelial growth of C. graminicola under five concentrations of essential oils (C1= 0,25 µL mL-1; C2= 0,50 µL mL-1; C3= 0,75 µL mL-1; C4= 1,0 µL mL-1; C5= 1,25 µL mL-1). In Test II, to assess the preventive effect, we used five types of oils (lemongrass, citronella, lemon balm, peppermint and neem) at four concentrations (2,5, 5,0, 7,5 e 10,0 µL mL-1). For curative effect, we used the same oils of preventive effect in two concentrations (2,5 e 5,0 µL mL-1). The essential oils of C. nardus and C. citratus were the most effective in reducing mycelial growth of C. graminicola (Test I). In Test II, the preventive effect, essential oils and concentrations significantly reduced the severity of the disease, especially oils of lemongrass, lemon balm and neem. In the curative effect, treatments reduced the severity, but no significant differences between them.

Effect of culture media and environmental factors on mycelial growth and pycnidial production of Lasiodiplodia theobromae in physic nut (Jatropha curcas ).

Physic nut (Jatropha curcas) is an important commercial bio-diesel plant species and is being advocated for development of waste and dry land. The collar and root rot caused by Lasiodiplodia theobromae is an important soil borne disease which causes considerable yield loss in this crop. In this study, the effects of culture media, temperature, photoperiod, carbon and nitrogen sources and pH on mycelial growth and pycnidial production were evaluated. Among the growth media tested, potato dextrose agar supported the highest growth followed by potato sucrose agar and corn meal agar. Among several carbon sources tested, carboxy methyl cellulose and sucrose were found superior for growth and pycnidial production. The nitrogen sources viz., ammonium oxalate and ammonium dihydrogen phosphate were recorded maximum mycelial growth and pycnidial production. The fungus grows at pH 5.0-9.0 and optimum growth was observed at pH 7.0.

Avaliação in vitro de diferentes métodos de análises de fungitoxicidade de óleos essenciais / Evaluation in vitro of different methods of analysis fungitoxicity of essential oils

Os problemas ambientais causados por fungicidas sintéticos têm elevado as buscas por métodos alternativos de controle de doenças de plantas. Objetivou-se avaliar o efeito do óleo essencial de capim citronela, sobre o fungo Rhizoctonia solani, em diferentes métodos de avaliação de fungitoxicidade in vitro. Foi utilizado delineamento inteiramente casualizado em esquema fatorial com quatro repetições, onde os fatores foram compostos por quatro métodos de avaliação da fungitoxicidade in vitro do óleo essencial (óleo essencial diluído em Tween 80 (0,5%) e incorporado ao meio de cultura BDA (batata, dextrose e ágar) ainda fundente; óleo essencial diluído em Tween 80 (0,5%) e distribuído na superfície do BDA; óleo essencial diluído em Tween 80 (0,5%) e distribuído em papel filtro fixado na superfície interna da tampa da placa de Petri; óleo essencial puro e distribuído na superfície do meio de cultura; e testemunha) e por cinco épocas de avaliação (2, 4, 6, 8 e 10 dias de incubação). Foram utilizados 0,25µL mL-1 do óleo do capim citronela em todos os tratamentos. Dos tratamentos avaliados o uso do óleo puro distribuído na superfície do meio de cultura foi mais eficiente na redução do diâmetro micelial em todas as avaliações. Neste método a taxa de crescimento micelial foi de 9, 02 mm dia-1, atingindo na última época de avaliação 79,77 mm.

Environmental problems caused by synthetic fungicides have increased the search for alternative methods of control of plant diseases. The objective was to evaluate the effect of essential oil of citronella grass, on the fungus Rhizoctonia solani, in different methods of in vitro fungitoxicity. We used a randomized design in a factorial design with four replications, where the factors were composed of four methods for assessing the in vitro fungitoxicity of the essential oil of citronella grass (essential oil diluted in Tween 80 (0.5%) and embedded in the culture medium PDA (potato dextrose agar) still melting, essential oil diluted in Tween 80 (0.5%) and distributed on the surface of the PDA; oil essential diluted in Tween 80 (0.5%) and distributed on filter paper attached to the inner surface of the lid of the Petri dish, pure essential oil and distributed on the surface of the culture medium, and control) and five evaluation periods (2, 4, 6, 8 and 10 days of incubation). Was used 0.25µL mL-1 of citronella oil in all treatments. Of the treatments evaluated the use of pure oil distributed on the surface of the culture medium was more effective in reducing the mycelial diameter in all evaluations. In this method the rate of mycelial growth was 9,02 mm day-1, reaching in last evaluation 79,77 mm.

Atividade antifúngica de óleo e extratos vegetais sobre Sclerotinia sclerotiorum / Antifungal activity of vegetable oils and extracts against Sclerotinia sclerotiorum

Considerando a importância do mofo branco, causado pelo patógeno Sclerotinia sclerotiorum na cultura da soja (Glycine max) e a falta de estudos sobre alternativas de controle deste patógeno, este trabalho teve como objetivo estudar o efeito de óleos e extratos vegetais sobre o crescimento micelial de S. sclerotiorum. No experimento de óleos essenciais, concentrações de 25, 50, 75 e 100 µg de i.a mL-1 de azadiractina, obtida de nim indiano (Azadirachta indica A. Juss), foram estudadas em associação às doses de 0, 1/3, 1/6, 1/8 e 1/10 do óleo de Karanja (Pongamia glabra). Quanto ao experimento de extratos vegetais, estudou-se as espécies Aroeirinha (Schinus molle L.), Mentrasto (Ageratum conyzoides L.), Alfavaca (Ocimum gratissimum L.), Losna (Artemisia absinthium L.), Jambolão (Syzygium cumini (L.) Skeels), Arruda (Ruta graveolens L.), Mandioca (Manihot esculenta Crantz), Santa Bárbara (Melia azedarach L.) e Pimenta longa (Piper aduncum L.) na concentração de 30%. Óleos e extratos vegetais foram incorporados, separadamente, ao meio de cultura BDA e vertido em placa de Petri de 9 cm de diâmetro, na qual depositou-se um disco de BDA, contendo micélio do fungo. As placas foram incubadas à temperatura de 22 ± 3 oC e fotoperíodo de 12 horas. As avaliações consistiram em medições diárias do diâmetro da colônia, durante 48 horas. A maior inibição do crescimento micelial foi diretamente proporcional ao aumento das concentrações dos óleos de nim indiano e de Karanja. A concentração de azadiractina, correspondente a 100 µg de i.a mL-1 com 1/3 de óleo de Karanja foi a mais eficiente na redução do crescimento micelial, com 63% de inibição. Em relação aos extratos vegetais, o fruto de pimenta longa foi o mais promissor sobre a redução do crescimento micelial, com 43% de inibição.

Considering the importance of white mold, caused by the pathogen Sclerotinia sclerotiorum, to soybeans (Glycine max) and the lack of studies about alternative control ways of plant pathogens, this study evaluated the effect of vegetable oils and extracts on the mycelial growth of S. sclerotiorum. Concentrations of 25, 50, 75 and 100 µg a.i. mL-1 of azadiractine, obtained from neem (Azadirachta indica A. Juss), were evaluated associated with the doses of 0, 1/3, 1/6, 1/8 or 1/10 of Karanja (Pongamia glabra) oil in the experiment with essential oils. Vegetable extracts of the species Schinus molle L., Ageratum conyzoides L., Ocimum gratissimum L., Artemisia absinthium L., Syzygium cumini (L.) Skeels, Ruta graveolens L., Manihot esculenta Crantz, Melia azedarach L. and Piper aduncum L. were evaluated in the concentration of 30%. Oils and vegetable extracts were incorporated, separately, into PDA culture medium and poured in 9-cm diameter petri plates, over which a PDA disk containing mycelium of the fungus was placed. The plates were incubated at 22 ± 3 oC and 12 hours lighting. The evaluations consisted of daily measurements of colony diameter for two days. The greatest mycelial growth inhibition was directly proportional to the concentration increase of neem and Karanja oils. Azadiractin concentration corresponding to 100 µg a.i. mL-1 with 1/3 Karanja oil was the most effective on reducing mycelial growth, with 63% inhibition. Among the vegetable extracts, fruits of long pepper were the most promising for reducing mycelial growth, with 43% inhibition.

Efeito da toxicidade de Cr (VI) e Zn (II) no crescimento do fungo filamentoso Aspergillus niger isolado de efluente industrial / Toxicity effect of Cr (VI) and Zn (II) on growth of filamentous fungi Aspergillus niger isolated from industrial effluent

Processos convencionais de tratamento de efluentes utilizam microrganismos vivos, o que sugere limitações relativas À toxicidade de metais para os microrganismos. O experimento consistiu em adicionar soluções monoelementares de Cr (VI) e Zn(II) em diferentes concentrações (0, 20, 50, 100, 200, 300, 400, 500 mg.L-1) ao meio de crescimento e observar a influência dos metais no crescimento micelial e germinativo do fungo Aspergillus Níger por verificação visual da expansão radial do micélio e da germinação de esporos, seguida de registro fotográfico. Os resultados mostraram que o metabolismo do fungo foi completamente inibido em concentrações acima de 500 mg Zn (II).L-1 e 150 mg Cr (VI).L-1. O ED50 (concentração de ingrediente ativo capaz de inibir 50 por cento do crescimento micelial do fungo) para os dois íons metálicos, nas condições estudadas, está na faixa entre 100 e 150 mg.L-1. Palavras-chave: metais pesados; inibição; crescimento micelial; Aspergillus niger; ED50.

Many standard processes of wastewater treatment use live microorganisms, which suggests limitations on a metal toxicity to the microorganism. The experiment consisted in adding mono elementary solutions of Cr (VI) and Zn (II) at different concentrations (0, 20, 50, 100, 200, 300, 400, 500 mg.L-1) to the growth mean, and to observe the influence of metals on mycelial and germinative growth of the Aspergillus niger fungus, by means of visual observation of the radial expansion of the mycelius and the germination of spores, followed by photograph registration. The results showed that the metabolism of the fungus was completely inhibited at concentrations above 500 mg Zn (II).L-1 and 150 mg Cr (VI).L-1. The ED50 (concentration of active ingredient capable of inhibiting 50 percent of mycelial growth of the fungus) for both metal ions, under the studied conditions, is in the range between 100 and 150 mg.L-1.

Antifungal activity of extracts from Brazilian Cerrado plants on Colletotrichum gloeosporioides and Corynespora cassiicola / Atividade antifúngica de extratos de plantas do Cerrado brasileiro sobre Colletotrichum gloeosporioides e Corynespora cassiicola

This study aimed to determine the antifungal activity of leaf aqueous and hydroethanolic extracts of 10 plants from the Brazilian Cerrado on Colletotrichum gloeosporioides and Corynespora cassiicola. Antifungal activity was measured through the incorporation of each extract in a culture media or spore suspension, at 50 percent concentration relative to the volume, determining respectively the mycelial growth and the spore germination. Then, the percentages of mycelial growth inhibition and spore germination inhibition were obtained based on the comparison with the control. The extracts had a variable action on the phytopathogens, from mycelial growth stimulation for Aristolochia esperanzae and Byrsonima verbascifolia extracts to complete inhibition of mycelial growth and spore germination for Myracrodruon urundeuva and Lafoensia pacari extracts. M. urundeuva, L. pacari and Caryocar brasiliense leaf extracts had antifungal activity against Colletotrichum gloeosporioides and Corynespora cassiicola; the hydroethanolic extracts presented more antifungal activity than the aqueous extracts, and spore germination of both phytopathogens was more affected than their mycelial growth.

O objetivo deste trabalho foi determinar a atividade antifúngica de extratos aquosos e extratos hidroetanólicos de folhas de 10 plantas do Cerrado brasileiro sobre Colletotrichum gloeosporioides e Corynespora cassiicola. A determinação da atividade antifúngica foi realizada pela incorporação do extrato em meio de cultura ou na suspensão de esporos, na concentração de 50 por cento em relação ao volume, determinando-se, respectivamente, o crescimento micelial e a germinação de esporos. Em seguida, pela comparação com a testemunha, foram obtidas as percentagens de inibição do crescimento micelial e da germinação dos esporos. Foi constatado comportamento variável dos extratos sobre os fitopatógenos, desde o estímulo no crescimento micelial para os extratos de Aristolochia esperanzae e Byrsonima verbascifolia, até a inibição completa do crescimento micelial e dagerminação dos esporos para os extratos de Myracrodruon urundeuva e Lafoensia pacari. Extratos de folhas de L. pacari, de M. urundeuva e de Caryocar brasiliense apresentaram atividade antifúngica sobre Colletotrichum gloeosporioides e Corynespora cassiicola; os extratos hidroetanólicos proporcionaram mais atividade antifúngica que os extratos aquosos, e a germinação de esporos de ambos os fitopatógenos foi mais afetada que o crescimento micelial.

Antagonistic and plant growth activity of Trichoderma isolates of Western Himalayas.

The genus Trichoderma is rapidly growing colonies bearing tufted or postulate, repeatedly branched conidiophores with lageniform phialides and hyaline or green conidia born in slimy heads. 62 isolates of Trichoderma species were isolated from different rhizospheric soil samples collected from different places located in Western Himalayas region. Out of these only two species were found i.e. Trichoderma hazianum and Trichoderma viride. Their efficacy against soil borne plant pathogens like Sclerotium rolfsii, Rhizoctonia solani and Sclerotinia sclerotiorum revealed that only three isolates amounting to 5% of the total collected isolates of this region were found highly antagonist. Among them 5% isolates were found against S. rolfsii, 13% isolates against R. solani, 10% against sclerotium caused above 80% inhibition of mycelial growth respectively. 6% isolates out of twenty seven utilized chitin by more than 80 and 16% isolates consumed cellulose by above 80% and therefore are producers of chitinase and cellulases. 58% isolates produced colonies having cottony texture and 41% produced dark green colonies. Pigmentation as observed from reverse side of the colony revealed that 70% of them did not produced pigment in the medium. Plant growth promotion measured as root and shoot lengths were significantly higher than in control. The maximum root length and shoot length were recorded when seeds were treated with isolates were recorded at Srinagar Garhwal was 4.70 and 4.75 cm out of all the isolates in which isolate recorded from Srinagar no 3 caused maximum percent seed germination which was significantly higher 79.49%.

Comparison of radial growth rate of the mutualistic fungus of atta sexdens rubropilosa forel in two culture media

In vitro culture of the mutualistic fungus of leaf-cutting ants is troublesome due to its low growth rate, which leads to storage problems and contaminants accumulation. This paper aims at comparing the radial growth rate of the mutualistic fungus of Atta sexdens rubropilosa Forel in two different culture media (Pagnocca B and MEA LP). Although total MEA LP radial growth was greater all along the bioassay, no significant difference was detected between growth efficiencies of the two media. Previous evidences of low growth rate for this fungus were confirmed. Since these data cannot point greater efficiency of one culture medium over the other, MEA LP medium is indicated for in vitro studies with this mutualistic fungus due its simpler composition and translucent color, making the analysis easier.

Mycelial Propagation and Molecular Phylogenetic Relationships of Commercially Cultivated Agrocybe cylindracea based on ITS Sequences and RAPD

This study evaluated the optimal vegetative growth conditions and molecular phylogenetic relationships of eleven strains of Agrocybe cylindracea collected from different ecological regions of Korea, China and Taiwan. The optimal temperature and pH for mycelial growth were observed at 25degrees C and 6. Potato dextrose agar and Hennerberg were the favorable media for vegetative growth, whereas glucose tryptone was unfavorable. Dextrin, maltose, and fructose were the most effective carbon sources. The most suitable nitrogen sources were arginine and glycine, whereas methionine, alanine, histidine, and urea were least effective for the mycelial propagation of A. cylindracea. The internal transcribed spacer (ITS) regions of rDNA were amplified using PCR. The sequence of ITS2 was more variable than that of ITS1, while the 5.8S sequences were identical. The reciprocal homologies of the ITS sequences ranged from 98 to 100%. The strains were also analyzed by random amplification of polymorphic DNA (RAPD) using 20 arbitrary primers. Fifteen primers efficiently amplified the genomic DNA. The average number of polymorphic bands observed per primer was 3.8. The numbers of amplified bands varied based on the primers and strains, with polymorphic fragments ranging from 0.1 to 2.9 kb. The results of RAPD analysis were similar to the ITS region sequences. The results revealed that RAPD and ITS techniques were well suited for detecting the genetic diversity of all A. cylindracea strains tested.

Cultural Conditions for Mycelial Growth and Molecular Phylogenetic Relationship in Different Wild Strains of Schizophyllum commune

The common split-gilled mushroom, Schizophyllum commune is found throughout the world on woody plants. This study was initiated to evaluate conditions for favorable vegetative growth and to determine molecular phylogenetic relationship in twelve different strains of S. commune. A suitable temperature for mycelial growth was obtained at 30degrees C. This mushroom grew well in acidic conditions and pH 5 was the most favorable. Hamada, glucose peptone, Hennerberg, potato dextrose agar and yeast malt extract were favorable media for growing mycelia, while Lilly and glucose tryptone were unfavorable. Dextrin was the best and lactose was the less effective carbon source. The most suitable nitrogen sources were calcium nitrate, glycine, and potassium nitrate, whereas ammonium phosphate and histidine were the least effective for the mycelial growth of S. commune. The genetic diversity of each strain was investigated in order to identify them. The internal transcribed spacer (ITS) regions of rDNA were amplified using PCR. The size of the ITS1 and ITS2 regions of rDNA from the different strains varied from 129 to 143 bp and 241 to 243 bp, respectively. The sequence of ITS1 was more variable than that of ITS2, while the 5.8S sequences were identical. A phylogenetic tree of the ITS region sequences indicated that the selected strains were classified into three clusters. The reciprocal homologies of the ITS region sequences ranged from 99 to 100%. The strains were also analyzed by random amplification of polymorphic DNA (RAPD) with 20 arbitrary primers. Twelve primers efficiently amplified the genomic DNA. The number of amplified bands varied depending on the primers used or the strains tested. The average number of polymorphic bands observed per primer was 4.5. The size of polymorphic fragments was obtained in the range of 0.2 to 2.3 kb. These results indicate that the RAPD technique is well suited for detecting the genetic diversity in the S. commune strains tested.