José Renato Stangarlin

Fungitoxicidade, atividade elicitora de fitoalexinas e proteção de pepino contra Colletotrichum lagenarium, pelo extrato aquoso de Eucalyptus citriodora

Secondary compounds present in medicinal plants can play important roles in plant-pathogen interactions, through immediate antimicrobial action or by inducing a defence mechanism in other plants that may be treated with such compounds. With the objective of verifying the potential of Eucalyptus citriodora in the alternative control of anthracnose in cucumber (Cucumus sativus), aqueous extract (AE) of this forest essence, autoclaved or unautoclaved, in concentrations of 0,1, 1, 5, 10, 15, 20 e 25% was used in the following experiments: induction of phytoalexins in sorghum (Sorghum bicolor) etiolated mesocotyls and soybean (Glycine max) cotyledons; fungitoxicity in vitro against Colletotrichum lagenarium conidia; local or systemic induction of resistance in cucumber plants. Water and Bion were used as control treatments. The results indicated that the autoclaved and unautoclaved AE induced phytoalexin synthesis in sorghum starting from the concentration of 1%. In soybean, gliceollin synthesis took place in 10% and 15% of the autoclaved and unautoclaved AEs, respectively. There was total inhibition in the spores germination and apressoria formation in concentrations of 20% and 1% of the autoclaved AE, respectively. As for the unautoclaved extract, there was 75% inhibition of the spores germination in 25% of AE and total inhibition in the apressoria formation in 15% of AE. Based on the parameter size of lesion, the unautoclaved aqueous extract of E. citriodora could potentially induce local resistance in cucumber against C. lagenarium.

Controle de Alternaria solani em tomateiro por extratos de Curcuma longa e curcumina: II. Avaliação in vivo

The discovery of plant secondary compounds with antimicrobial activity is very promising. Turmeric, Curcuma longa, has compounds in its rhizomes with fungicidal activity. The objective of this study was to evaluate the in vitro fungitoxic activity of turmeric extracts and curcumin against Alternaria solani. Four different concentrations (0, 1, 5, 10 and 20%) of aqueous extracts of turmeric rhizomes (sterilized by autoclave) and four curcumin solutions (0, 50, 100, 200 and 400 mg/L) were incorporated into potato dextrose agar medium in order to evaluate fungal mycelial growth and sporulation. To evaluate the effect of using an autoclave, 10% and 15% turmeric extracts were sterilized by filtration. The effects of autoclaved and non-autoclaved turmeric extracts and curcumin on in vitro spore germination were tested. The concentrations of 10 and 15% of non-autoclaved turmeric extracts inhibited the mycelial growth by 38.2% and 23.2% respectively and the fungal sporulation by 71.7% and 87% respectively. When turmeric extracts were autoclaved, neither mycelial growth nor spore germination was inhibited and the effect on sporulation was reduced, suggesting the presence of thermolabile antimicrobial compounds. The non-autoclaved 5% extract inhibited spore germination by up to 15%. At the highest concentration, the curcumin solution inhibited mycelial growth by 29.5%. Neither in vitro sporulation nor spore germination was affected by curcumin. These results show the fungitoxic potential of turmeric and curcumin against A. solani.

Atividade antifúngica e proteção do tomateiro por extratos de plantas medicinais

The present work aimed to evaluate, in vitro, the fungitoxicity of aqueous crude extracts (ACEs) of Achillea millefolium, Artemisia camphorata, Cymbopogon citratus and Rosmarinus officinalis to Alternaria solani and their protective effect on tomato plants under greenhouse conditions. To evaluate the antifungal activity of ACEs, they were incorporated into potato-dextrose-agar medium and the inhibition of mycelial growth, sporulation and conidia germination was evaluated. The protective effect in plants was evaluated by means of preventive spraying (72 hours before inoculation) with ACE, at concentrations of 10 and 20%, in the first two leaves. Disease severity was verified 15 days after inoculation. It was observed that ACEs did not inhibit mycelial growth, but they had significant effects on the reduction of germination and sporulation, especially the ACEs of A. camphorata, C. citratus and R. officinalis, at concentrations up to 20%. A reduction was observed in the number of lesions in relation to the control, in leaves above those treated, observing the systemic effect of the extracts. The extracts from the studied plants may be promising in the control of early blight on tomato plants.

Fungitoxicidade, atividade elicitora de fitoalexinas e proteção de alface em sistema de cultivo orgânico contra Sclerotinia sclerotiorum pelo extrato de gengibre

Sclerotinia sclerotiorum is a soilborne fungus and causes white mold, sclerotinia cottony rot in lettuce (Lactuca sativa) and other crops. The control of this disease is very difficult, since that fungus produces resistance structures, the sclerodia. In the search of new methods for disease control, the plant extracts with therapeutical properties arise as a new option. The effect of aqueous crude extract (ACE) of ginger (Zingiber officinalis) was evaluated in vitro at concentrations of 1, 5, 10, 15, 20 and 25% on S. sclerotiorum mycelial growth and sclerodia production. The efficiency of ginger on the production of lettuce plants was also verified in plants and inoculated with the pathogen. Disease incidence, crop yield and peroxidase activity were analyzed in plant tissue. Water and the resistance inducer agent acibenzolar-S-methyl were used as control. Additionally, the ability of ACE in inducing accumulation of phytoalexins 3-deoxyanthocyanidin and glyceollin was evaluated in sorghum and soybean bioassays, respectively. The results showed the antimicrobial activity of ginger on the inhibition of mycelial growth and sclerodia production. In the lettuce crop, it was observed that the application of ginger mass on the soil close to plants basis increased of peroxidase activity and reduced disease incidence. The presence of elicitor compounds in the ACE was detected by the production of phytoalexins in sorghum and soybean, with dose-dependent responses. These results showed the potential of Z. officinalis on the control of S. sclerotiorum in lettuce that can occur by antimicrobial activity and defense mechanisms induction.

Contribuição ao estudo das atividades antifúngica e elicitora de fitoalexinas em sorgo e soja por eucalipto (Eucalyptus citriodora)

Secondary compounds found in medicinal plants play important role in plant-pathogen interactions by having direct antimicrobian effect on them or inducing the defense mechanisms on defense of other plants. To verify the fungi toxic effect of the eucalyptus on the micelial growth of Rhizoctonia solani, Sclerotium rolfsii, Phytophthora sp, Alternaria alternata and Colletotrichum sublineolum, the crude extract (CE) was incorporate to PDA and the essential oil (EO) and distributed in the surface of the middle with Drigalski loop. Germination of spores of C. sublineolum was also evaluated in the presence of different brackets of EO using two methodologies. In order to verify the induction phytoalexins sorghum mesocotyls were asperged with CE to 20% or then dived in suspensions of EO. For the gliceolin induction, 20 µL of CE were put in cotyledons soybean. The presence of fungitoxic compounds in EO and CE through the thin layer chromatography was also evaluated. The results evidenced inhibition of the fungus micelial growth for concentrations of CE above 20%. All of the aliquots of the oil inhibited micelial growth micelial fungi, except for R. solani whose inhibition happened for aliquots above 20µL. There was inhibition of 100% in conidia germination for all of the aliquots of EO tested in the first methodology. However, in the second methodology EO did not promote inhibition of conidia germination. It was observed the presence of one fraction fungitoxic in EO, and CE did not present fungitoxic fractions. There was the phytoalexins production just in sorghum mesocotyls treated with CE.

Induction of resistance in beans against Pseudocercospora griseola by culture filtrates of Pycnoporus sanguineus

With the aim of developing an alternative method for the control of angular leaf spot of common bean, caused by Pseudocercospora griseola, aqueous extracts of Pycnoporus sanguineus culture filtrates were evaluated in vitro for antimicrobial activity against P. griseola, and possible induction of resistance in bean against P. griseola. The role of the defense enzymes peroxidase, polyphenol oxidase, and β-1,3-glucanase, and the content of proteins and chlorophyll, were also investigated. For the in vitro and greenhouse experiments the culture filtrate of P. sanguineus at the concentrations of 1 to 20% was used, and water, acibenzolar-S-methyl (ASM: 75 mg i.a. L-1) and fungicide (azoxystrobin: 40 mg i.a. L-1) served as control treatments. In the in vitro study, culture filtrate did not inhibit mycelial growth, sporulation and conidia germination of P. griseola. The total area under the angular leaf spot progress curve was reduced by 82 and 49% respectively, for greenhouse and field experiments in relation to the water control. The activities of peroxidase and polyphenol oxidase, and the content of proteins and chlorophylls, increased in plants treated with the extract. The results from this study indicate the potential of P. sanguineus culture filtrate as an alternative control for angular leaf spot on beans.

Induction of plant defense responses by Ocimum gratissimum L. (Lamiaceae) leaf extracts

Aqueous extracts of the leaves of Ocimum gratissimum at 10, 25, 40 and 50% (w/v) concentrations induced the production of phytoalexins in soybean cotyledons and sorghum mesocotyls. The aqueous extracts also induced systemic resistance in cucumber to Colletotrichum lagenarium, reflected by reduction in disease incidence and an increase in chitinase production. Modes of action and the existence of possible elicitors of defense response in O. gratissimum leaf extracts are discussed.

Resistance induction in bean plants against angular leaf spot by extracts from Pycnoporus sanguineus mycelium

Angular leaf spot, caused by the fungus Pseudocercospora griseola, is a major disease of common bean (Phaseolus vulgaris L.) in Brazil. The objective of this research was to develop an alternative method for controlling this disease based on an aqueous extract of Pycnoporus sanguineus mycelium. It was evaluated the antimicrobial activity of the aqueous extract against P. griseola as well as its activity of resistance induction against angular leaf spot. The role of the plant defense enzymes peroxidase, polyphenol oxidase and β-1,3-glucanase, and the proteins and chlorophyll content was investigated. The experiments in vitro and in greenhouse used extracts from mycelium of P. sanguineus and water, Acibenzolar-S-methyl (ASM: 75 mg/L) and the fungicide azoxystrobin (40 mg/L) were the control treatments. In vitro, the extract of mycelium inhibited the mycelial growth as well as the sporulation and conidia germination of P. griseola. However severity was reduced in 93% and 50% in greenhouse and field respectively. The activities of peroxidase, polyphenol oxidase and the content of proteins and chlorophylls had an increment in plants treated with extract. These results indicate the potential of P. sanguineus for alternative control of the angular leaf spot in bean.

Caracterização parcial de frações obtidas de extratos de Cymbopogon nardus com atividade elicitora de fitoalexinas em sorgo e soja e efeito sobre Colletotrichum lagenarium

Medicinal plants present potential to control phytopathogens due to the direct effect or by the activation of defense mechanisms like phytoalexins. Then, the present work had the objective to verify the antifungal and elicitor effect of citronella (Cymbopogon nardus) obtained from methanolic and ethanolic extracts. It was studied the accumulation of phytoalexins in the mesocotyls of sorghum and in cotyledon of soybean, in response to application of fractions from crude extracts methanolic (EME) and ethanolic extracts (EET) of citronella. The EME and EET extracts were fractioned by gel filtration chromatography (CFG) and three fractions were obtained from the methanolic extract (FMI, FMII and FMIII), and two fractions from the ethanolic extract (FEI and FEII). The molecular weight for the FMI, FMII, FMIII, FEI and FEII were 69.29, 40.51, 18.72, 65.89 and 24.11 kDa, respectively. The fractions obtained from the CFG were used in a bioassay of germination of C. lagenarium spores and of induction of phytoalexins in the mesocotyls of sorghum and cotyledon of soybean. There was no significative effect of the fractions EME and EET on germination and appressorium formation by the pathogen. In the accumulation of phytoalexins in soybean cotyledons there was no significant effect of the fractions FMI, FMII, FMIII, FEI and FEII. However, it was detected a significative effect of fractions obtained by CFG on the production of phytoalexins in mesocotyls of sorghum. The highest accumulation of phytoalexin in mesocotyls of sorghum was obtained in the FMI and FMIII fractions, which differed significantly to the control. Based on the obtained results, it is possible to conclude that fractions partially purified obtained from EME of C. nardus present potential to induce phytoalexins in sorghum mesocotyls.

Differential occurrence of the oxidative burst and the activity of defence-related enzymes in compatible and incompatible tomato-Oidium neolycopersici interactions

The generation and accumulation of hydrogen peroxide (H2O2) and superoxide anion (O2.-), as well as guaiacol peroxidase, catalase, polyphenol oxidase, β-1,3-glucanase and chitinase activity, were studied in leaves of resistant and susceptible tomato genotypes inoculated with Oidium neolycopersici. Plants of the resistant genotype CNPH 1287 (Solanum habrochaites sin. Lycopersicon hirsutum) and susceptible genotype Santa Cruz Kada (S. lycopersicum sin. Lycopersicon esculentum), with the seven-nine and five-seven leaves completely developed, respectively, were inoculated in the second, third and fourth true leaves. Leaves were collected at the time of inoculation and at 4, 8, 12, 24, 48, 72, 96 and 120 h post inoculation (hpi). The production and accumulation of H2O2 and O2.- were evaluated in situ using diaminobenzidine and nitroblue tetrazolium, respectively. Starting at 24–48 hpi, high accumulation of H2O2 and O2.- was detected, and epidermal cells demonstrated a hypersensitive response, especially in the inoculated leaves of the resistant plant (S. habrochaites). An increase in guaiacol peroxidase, catalase, polyphenol oxidase, β-1,3-glucanase and chitinase activity was mainly detected by 24 hpi in the resistant plant. An association between the production of reactive oxygen species and the activity of enzymes related to reactive oxygen species metabolism (guaiacol peroxidase, catalase), hydrolytic enzymes (β-1,3-glucanase and chitinase) and phenol metabolism enzymes (polyphenol oxidase), as well as hypersensitive response, was evident during the defence responses of the resistant plants when inoculated with O. neolycopersici.