Control of plant diseases using extracts from medicinal plants and fungi

Transcription

1 Science against microbial pathogens: communicating current research and technological advances Control of plant diseases using extracts from medicinal plants and fungi J. R. Stangarlin1, O. J. Kuhn1, L. Assi1, K. R. F. Schwan-Estrada2 1 Western Paraná State University UNIOESTE, Rua Pernambuco 1777, Caixa Postal 91, CEP , Marechal Cândido Rondon, Paraná, Brazil. jrstangarlin@pq.cnpq.br 2 Maringá State University UEM, Av. Colombo 5790, CEP , Maringá, Paraná, Brazil Actually, with the objective to find new technologies, ecologically or environmentally safer, for the control of plant diseases, mainly in organic growth, alternative methods for the control of phytopathogens are been development. This kind of alternative methods are been investigated in our Biological and Alternative Control of Plant Diseases research group. An overview of the our researches using medicinal plants and fungal extracts for the plant disease control in the last 10 years and examples of in vitro and field essays are presented. Keywords resistance induction; alternative control; botanical formulation; organic growth 1. Introduction The intensive and indiscriminate use of pesticides in agriculture has caused many problems to the environment such as water, soil, animals and food contamination; poisoning of farmers; elimination of non-target organisms; and selection of phytopathogens, pest and weed insensitive to certain active ingredients [1]. Aiming to minimize the negative effects of pesticides are been development the alternative control of plant disease, which includes the biological control, the induction of resistance and the use of natural products with induction of resistance and/or with direct antimicrobial activities. In the latter include the use of extracts and essential oils from medicinal plants [1, 2], as well as the extracts obtained from basidiomycetes [3, 4, 5, 6, 7]. The biological control is defined as the use of antagonistic organisms for the control of microorganisms, reducing the amount of inoculum that determines the extent of disease [8]. The induction of resistance promotes the activation of the latent plant defense systems, which manifests itself when it comes into contact with a biotic [9, 10, 11, 12, 13] or abiotic elicitor [14, 15]. The expression resistance of induction both can be used to denote local protection, this is, the induction of a resistance only in the tissues in which it was applied the treatment with the inducing agent, also can indicate a systemic resistance that manifest far from the tissue where the elicitor was applied [16]. 2. Medicinal plant extracts Plant extracts used to control of the phytopathogens have been obtained mainly from tree species such as eucalyptus and neem (24% of the studies with extracts) and herbaceous species like garlic, citronella, mint, rue, yarrow, ginger, basil, camphor, turmeric and ocimum (54%). Besides these there are other 237 plants from the Brazilian flora whose antimicrobial potential was tested by Brazilian researchers. With respect to groups of pathogens, the majority of the work is with those that cause disease in the plant canopy (30% of the works with extracts), like the genus Alternaria, Bipolaris, Crinipellis, Corynespora and Colletotrichum, which respond alone for 15% of the works. The soil-born pathogens represent 20% of the researches, especially Rhizoctonia, Sclerotium, Sclerotinia, Fusarium and Phytophthora. Post-harvest pathogens like Penicillium, Aspergillus and Rhizopus are in 9% of the works and Meloidogyne nematode in 9.5%. For the host plants, 30% of the work are with crops like beans, soybeans, coffee, wheat, cotton and cassava; 20% with vegetables like cucumber and tomato, this later representing alone 15% of all the researches with extract; and 10% with the fruits like papaya, strawberry and cocoa. Details about these data can be found in the review of Stangarlin and colleagues [11]. From these presented numbers we can have an overview of the researches on alternative control of disease in plants in Brazil using natural extracts. Besides that, other researches have been made to identify plants whose extracts have compounds with biological action against phytopathogens or that induce plant resistance. Some of these works are detailed below, focusing mainly the results published of the research group Biological and Alternative Control of Plant Diseases - COBALFI (Western Paraná State University - UNIOESTE campus Marechal Cândido Rondon, Brazil). 2.1 Artemisia camphorata (camphor) Aqueous extract (AE) of camphor was used to evaluate the fungitoxic activity against Bipolaris sorokiniana and the induction of resistance to black point wheat. The AE in concentrations of 1, 5, 10, 15, 20, 25 and 50% was prepared with antioxidant (Na2SO3 0.25%) or not, and autoclaved or not autoclaved. To evaluate induction of resistance against 1033

2 A. Méndez-Vilas (Ed.) black point disease, wheat plants with 30 days were sprayed with AE. At 24, 48 and 72 h after the treatments, the leaves were inoculated with the pathogen (1x10 4 conidia/ml). The AE 50% caused 39% of mycelial growth inhibition. The AE 10% totally inhibited the fungus sporulation in vitro. The AE not autoclaved inhibited only 20% of the conidia germination but the autoclaved AE did not cause inhibition showing the presence of thermo-sensible compounds. The incorporation of antioxidant Na 2 SO 3 in the AE increased the inhibition of conidia germination. The AE of camphor also induced systemic resistance in wheat plants, reducing the size (up to 29%) and the number of spots (up to 60%). The induction was higher in plants treated with camphor 72 h before the inoculation with pathogen [17]. To evaluate the post-harvest control of anthracnose in banana, an in vitro assay was conduced to measured the mycelial growth of Colletotrichum musae in solid and liquid media containing 1, 5, 10, 15, 20 and 25% of camphor (fresh weigh/volume). To in vivo assay was evaluated the post-harvest control by immersing banana fruits for 3 min in the camphor extracts, using as control treatments Benomyl (0.25 g/l) and distillated water. The results showed that camphor inhibited 41% the mycelial growth and 86% the sporulation, in solid medium, although had stimulated the mycelial growth in liquid medium. The severity of anthracnose was reduced to 67% and 56% for camphor and fungicide treatments, respectively [18]. 2.2 Curcuma longa (turmeric) Control of Xanthomonas axonopodis pv. manihotis The control of cassava bacterial wilt was evaluated in vitro by using aqueous extract of four turmeric genotypes from Jaboticabal-SP, Mara Rosa-GO, Maringá-PR and Mercedes-PR, as well as in vivo, by treatment of infected cassava stems and their cultivation at field conditions. The results showed that in vitro experiment, turmeric extract inhibited completely the bacteria growth in the concentration of 10% for the genotype from Mercedes, while for the Jaboticabal s turmeric there was a total control at 15% and for Mara Rosa at 20%. Turmeric genotype from Maringá not showed full inhibition of the bacterial growth in none of the extract concentrations used. At field conditions, sproutings were extremely low, due to the stems infection degree. Turmeric extract at 10%, from Mercedes, was harmful for the cassava, reducing stand regarding the treatments control. Possibly there was a direct toxic action on the cassava physiology or susceptibility induction. But in the concentration of 1% of turmeric from Maringá, there was no statistical difference in relation to the control treatment for the stand of plants parameter. The chemical control was not completely efficient. There was no statistical difference among treatments for both disease severity and cassava productivity [19] Control of Alternaria solani Four different concentrations (0, 1, 5, 10 and 20%) of aqueous extracts of turmeric rhizomes (sterilized in an autoclave) and four curcumin solutions (0, 50, 100, 200 and 400 mg/l) were incorporated into potato dextrose agar medium in order to evaluate mycelial growth and sporulation of Alternaria solani. The concentrations of 10 and 15% of turmeric extracts inhibited the micelial growth by 38.2% and 23.2% respectively, and the fungal sporulation by 71.7% and 87% respectively. At the highest concentration, the curcumin solution inhibited the mycelial growth by 29.5%. Neither in vitro spore production nor spore germination was affected by curcumin [20]. To evaluate the control of tomato early blight at greenhouse conditions were used 1 and 10% turmeric extracts, 50 and 100 mg/l curcumin solutions, acibenzolar-s-methyl (ASM) (25 mg active ingredient a.i/l), copper oxychloride (1,100 mg a.i./l), azoxystrobin (80 mg a.i./l) and control (water). The disease control showed by turmeric extracts and curcumin solutions was similar to cupric fungicide, but inferior to azoxystrobin control. There was no statistical difference in commercial fruit production between treatments. Only the treatment with 50 mg/l of curcumin had higher percentage of bigger fruits compared to the control [21]. 2.3 Eucalyptus citriodora (eucalyptus) Aqueous extract (AE) of eucalyptus in concentrations of 0.1, 1, 5, 10, 15, 20 and 25% was used in the following experiments: induction of phytoalexins in sorghum etiolated mesocotyls and soybean cotyledons; in vitro fungitoxicity against Colletotrichum lagenarium; and local or systemic induction of resistance in cucumber plants against anthracnose. Water and acibenzolar-s-methyl were used as control treatments. The eucalyptus extract induced phytoalexin synthesis in sorghum starting from the concentration of 1%. In soybean, glyceollin synthesis took place from 10% of aqueous extract. The phytoalexin synthesis in different incubation periods showed that greater inductions take place from 48 hours on. There was total inhibition in the spores germination and apressoria formation in concentrations of 20%. Based on the parameter size of lesion, the aqueous extract of E. citriodora was capable to induce local resistance in cucumber plants against C. lagenarium [22]. 1034

3 Aqueous extract from E. citriodora fresh leaves, in concentrations up 20%, were efficient to inhibited in 100% the mycelial growth of Colletotrichum sublineolum, Phytophthora sp. and Sclerotium rolfsii, in 75% to Rhizoctonia solani and in 45% to Alternaria alternate, under in vitro assays [23]. 2.4 Zingiber officinale (ginger) The effect of aqueous extract of ginger was evaluated at concentrations of 1, 5, 10, 15, 20 and 25% on Sclerotinia sclerotiorum mycelial growth and sclerodia production, in vitro. The efficiency of protection of ginger was also verified in lettuce plants growth organically and inoculated with the pathogen. Besides the disease incidence, the crop yield and the peroxidase induction were analyzed in the tissue plants. Water and the resistance inductor acibenzolar-s-methyl were used as control treatments. Additionally, the elicitor ability of ginger extract in inducing accumulation of the phytoalexins 3-deoxyanthocyanidin and glyceollin was evaluated in sorghum and soybean bioassays, respectively. The results showed the antimicrobial activity of ginger with mycelial growth and sclerodia production inhibition. In the lettuce crop, it was observed that the application of ginger mass on the soil near to plants basis caused a bigger activity of peroxidase enzyme and the reduction of disease incidence. The presence of elicitor compounds in the ginger extract was detected by the production of phytoalexins in sorghum and soybean, with dose-dependent response [24]. 2.5 Ocimum gratissimum (wild basil) The in vitro effect of wild basil crude extract (CE) (autoclaved or sterilized by filtration) as well as the iprodione + thiram fungicide on the mycelial growth and sporulation of Bipolaris sorokiniana was evaluated. Besides, wheat seeds naturally infected with B. sorokiniana were submitted to the following treatments: 1) fumigation with essential oil; 2) immersion with autoclaved CE and; 3) non autoclaved CE at different concentrations (1, 5, 10, 15, 20, 25, and 50%; w/v); 4) sprinkling with dry leaves of the medicinal plant and with the fungicide. The autoclaved CE showed, even partially, higher inhibition of mycelial growth in all the tested concentrations than CE sterilized by filtration. In greenhouse, the fumigated seeds (7 days) showed the same emergency speed (EV) concerning to the control (water). There were no statistics differences among the analyzed parameters (EV, emergency percent, height, dry mass, fresh mass, and transmission rate (TR)) when the treatment by immersion was compared with the no treated control. Seeds treated with the dry powder of medicinal leaves did not differ statistically from the chemical control, when the same parameters were evaluated, but presented higher TR of the pathogen. In the laboratory, seeds submitted to the fumigation process presented a higher number of health seeds than the non treated control, while the treatments by immersion in the CE and sprinkling with dry leaves did not differ concerning to the same parameter [25]. 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 against Colletotrichum lagenarium, reflected by reduction in disease incidence and an increase in chitinase production [26]. The essential oil and crude extract of wild basil, harvested in different seasons, were tested in vitro on the mycelial growth of Rhizoctonia solani, Sclerotium rolfsii, Phytophthora sp. and Alternaria alternata. To evaluate the effect of the essential oil, sterilized oil aliquots of 20 µl, 40 µl and 60 µl were distributed on the surface of potato-dextroseagar (PDA). The aqueous extracts were filtrated and incorporated in PDA at 1%, 5%, 10%, 15%, 20%, 25% and 50%. The results showed total inhibition of mycelial growth in the different aliquots of essential oil. The extract collected from plants in autumn had better results; in this season the aqueous extract at 5% was enough to provide inhibition of 100% on mycelial growth of A. alternata and S. rolfsii [27]. 2.6 Tagetes patula (marigold) Aqueous extract of flowers, leaves and roots of Tagetes patula were obtained by infusion in the proportion of 50 grams of dehydrated material in 1,000 ml of distilled water and tested in vitro on eggs and second-stage juveniles (J2) of Meloidogyne incognita, without dilution and diluted 1:1, 1:2, 1:3 and 1:4 (extract:water, v:v), and in plants of tomato cv. Santa Cruz Kadá cultivated in pots, without dilution and diluted 1:1. For in vivo assays, the extract was sprayed on the leaves, soil or both by four ways: weekly during eight weeks; at the transplanting (one week before inoculation); and together or one week after inoculation. The extract of flowers, leaves and roots inhibited the hatching, the mobility, and caused mortality of J2 in vitro. Greater nematicide effect was obtained with roots extract, which promoted mortality up to 68% of J2. In tomato plants, results were not satisfactory with only one application of Tagetes extract. Weekly applications promoted plant development and decreased galling and the nematode population. Greater effect was obtained by flower extract application, followed by leaves extract. Slight or non effect was observed by root aqueous extract. Flower extract without dilution inhibited galling up to 62.2% and decreased 61.5 and 52.8% the number of J2 in the soil and eggs in the roots, respectively. Applications on the soil or leaves presented similar results. These results demonstrated the potential of T. patula extract, mainly of flowers, to protect tomato plants against M. incognita. Furthermore, it was hypothesized that induced resistance was involved in the responses observed [28]. 1035

4 A. Méndez-Vilas (Ed.) 2.7 Rosmarinus officinalis (rosemary) and Lippia alba (bushy lippia) Crude extract of L. alba and R. officinalis, alone or in combination, at concentration of 5, 10, 15, 20, 25 e 50%, sterilized by autoclaving, were used to verify the effect over the mycelial growth of Colletotrichum graminicola, Alternaria alternata, Phytophthora sp., Rhizoctonia solani and Sclerotium rolfsii. The test in vitro indicated that in relation to the crude extract of rosemary, there was a bigger inhibition in concentrations of 50% for all the fungi, except to A. alternata, which the inhibition was bigger in concentrations of 10 e 15%. With relation to the crude extract of lippia, the fungi S. rolfsii, A. alternata and R. solani presented inhibition to mycelial growth in concentration of 50%, what did not occur with C. graminicola, for which the best results were in concentrations above of 10%, and of Phytophthora sp., where the inhibition occurred in concentrations of 15%. When in the presence of rosemary and lippia extracts mixture, the results obtained showed that occurred inhibition for the mycelial growth of C. graminicola, A. alternata and S. rolfsii in concentration of 50%, while that for R. solani and Phytophthora sp. the inhibition was bigger in concentrations of 25 e 15%, respectively [29]. 2.8 Achillea millefolium (yarrow) e Artemisia absinthium (absinthium) The effect of aqueous extract of A. millefolium and A. absinthium, isolated or in mixture, in concentrations of 5, 10, 15, 20, 25 e 50%, was verified on the mycelial growth of fungi Alternaria sp. (isolated from coffee leaves), Alternaria alternata, A. steviae and A. solani. In the presence of crude extract of yarrow there was no inhibition of mycelial growth of all fungi isolates. The crude extract of absinthium did not inhibit the mycelial growth of A. solani and Alternaria sp. To A. steviae the major inhibition occurred in concentration of 50%. A. alternata presented about 20% of reduction in the mycelial growth for all the tested concentrations. To the mixture of plant extracts, the inhibition was smaller than when it was used the extracts separately, but with no inhibitory effect in the mycelial growth of A. solani. For the other fungi isolates the major inhibition of mycelial growth was around of 20% [30]. The fungitoxicity of aqueous extracts of Achillea millefolium, Artemisia camphorata, Cymbopogon citratus and Rosmarinus officinalis was evaluated against Alternaria solani, as well their protective effect on tomato plants under greenhouse conditions. To evaluate the antifungal activity of extracts, 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 plant extracts, at concentrations of 10 and 20%, in the first two leaves. Disease severity was verified 15 days after inoculation. It was observed that extracts did not inhibit mycelial growth, but they had significant effects on the reduction of germination and sporulation, especially the extract 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 [31]. The antimicrobial effect of aqueous extracts and essential oil of Achillea millefolium on mycelial growth, sporulation and spores germination of Corynespora cassiicola, as well as peroxidase activity in cucumber plants treated with those extracts were evaluated. Crude extract (1; 5; 10; 20 and 25%) was incorporated into potato-dextrose-agar medium (PDA), and essential oil (20, 40, 60, 100 and 200 µl) was distributed over the surface of the PDA. The mycelial growth was measured daily for 7 days. In the in vivo test, cucumber plants were sprayed with aqueous extracts at concentrations of 1, 10 and 25%, 4 and 2 days before inoculation and at the same time of inoculation with C. cassiicola. The disease severity and peroxidase activity were evaluated. For crude extract, neither mycelial growth nor spore germination and sporulation was inhibited at any of the tested concentrations. In the treatments with essential oil there was inhibition up to 63% of the mycelial growth. In the spore production and spores germination the inhibition was 100% and 98%, respectively, for 200 µl of essential oil. In the control of the disease the most effective treatment was the aqueous extract 25% when carried out at the same time as the inoculation. In the evaluated period, no peroxidase activity was found [32]. 2.9 Cymbopogon nardus (citronella) 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 methanol-extracts (EME) and ethanol-extracts (EET) of citronella. The EME and EET extracts were fractioned by gel filtration chromatography (CFG) and three fractions were obtained from the methanol-extract (FM I, FM II and FM III), and two fractions from the ethanol-extract (FE I and FE II). The molecular weight for the FM I, FM II, FM III, FE I and FE II were 69.29, 40.51, 18.72, and kda, respectively. The fractions obtained from the CFG were used in a bioassay of germination of Colletotrichum lagenarium spores and of induction of phytoalexins in the mesocotyls of sorghum and cotyledon of soybean. There was no statistical difference to 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 FM I, FM II, FM III, FE I and FE II. However, it was detected a significant 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 FM I and FM III fractions, which differed significantly to the control [33]. 1036

5 Science against microbial pathogens: communicating current research and technological advances 2.10 Bidens pilosa (spanish needle), Thymus vulgaris (common thyme) The fungitoxicity of aqueous extracts of B. pilosa, T. vulgaris, L. alba and R. officinalis was evaluated in vitro on development of Alternaria alternata, Colletotrichum graminicola, Rhizoctonia solani and Sclerotium rolfsii. The extract were incorporated to potato-dextrose-agar alone or in mixture, at 0, 1, 5, 10, 15, 25 and 50% concentrations. Measurement in fungal colonies was taken daily until the control treatment covered 2/3 of the culture media. The mixtures of T. vulgaris with B. pilosa did not have positive effect because the extract of B. pilosa did not reduce the fungal growth of R. solani and in mixture inhibited the effect of T. vulgaris extract. However, the mixture of extracts of R. officinalis and L. alba produced better results than isolated extracts, with 60% of growth inhibition of A. alternata. The extract of T. vulgaris inhibited the growth of C. graminicola at 97% [34] Cymbopogon citratus (lemongrass) and Cymbopogon martinii (palmarosa) Crude extracts of C. citratus and C. martinii were used to verify the in vitro fungitoxic potential against Colletotrichum sp. in bell pepper, in post-harvest conditions. To in vitro assay, concentrations of 1, 5, 10, 15, 20, 25 e 50% of the crude extract (CE) were incorporated into PDA and aliquots of 1, 20, 40 and 60 μl of the essential oil (EO) were distributed on PDA surface to evaluate micelial growth. It was evaluated sporulation for the counting of spores in hemacytometer, and the spores germination and appressorium formation in ELISA plates. To in vivo assay, bell pepper fruits were inoculated with spores suspension (1x105 spores/ml) and after 2 days treated with 10 and 20% extract prepared for microwaves irradiation. Distilled water and citric extract were used as controls. Fruits weight reduction and disease severity were evaluated. The CE of the two species not significantly inhibited the micelial growth, the sporulation and the spores germination, however it had inhibitory effect on the appressorium formation. The essential oil presented inhibitory effect on the mycelial growth, sporulation, spores germination and appressorium formation. To in vivo assay, the disease was not controlled for the CE. In the parameter fruits weight reduction, the CE was similar to the positive control, however smaller fruits weight reduction was verified with C. martinii CE at concentration of 10% [35] Adiantum capillus-veneris (maidenhair fern) Derivatives of Adiantum capillus-veneris, obtained from the powder of shadow-dried maidenhair fern leaves, were prepared by infusion, maceration and decoction, autoclaved and non-autoclaved, in the concentration of 0.1%, 0.25%, 0.5%, 1%, 1.25%, 2.5%, 5%, 10%, and 20%. The germination of Phakopsora euvitis and Pseudocercospora vitis spores, the mycelial growth of Colletotrichum musae and Sclerotium rolfsii, and the multiplication of Erwinia sp. and Bacillus subtilis bacteria were evaluated. The extract obtained by non-autoclaved maceration presented the best effect against P. euvitis, with reduction of 75% on uredospores germination in the concentration of 20%. Against P. vitis, the derivative obtained by non-autoclaved maceration was more effective, presenting 99% of reduction on conidia germination in the concentration of 20%. The smallest mycelial growth of C. musae and S. rolfsii was obtained by autoclaved maceration with 39% and 83% reduction, respectively. For Erwinia sp., the greatest inhibitory effect was obtained with the 25% autoclaved maceration that inhibited the bacterial growth at 99%. For B. subtilis, there was no significant inhibition [36] Mikania glomerata (guaco) Alcoholic extract 50º GL of M. glomerata was evaluated regarding to: in vitro antimicrobial activity through bacterial growth in 100, 250, 500 and 1000 mg/l of the alcoholic extract; induction of local or systemic resistance in 25 days old cauliflower, with spray of alcoholic extract concomitantly and three days before the inoculation with the pathogen (water and Bordeaux mixture were used as control); peroxidases activity in leaves of cauliflower treated and not treated, and harvested concomitantly, 24, 48 and 72 hours after spraying the alcoholic extract and also after inoculation. The alcoholic extract of M. glomerata in vitro showed inhibition of the bacterial growth in the concentrations of 250, 500 and 1000 mg/l. The concentrations of 500 mg/l and 1000 mg/l inhibited 24% and 38% the bacterial growth. This inhibition could be due to antibacterial compounds in the alcoholic extract. An inhibition of the disease in vivo occurred only in the leaves treated with 100 and 500 mg/l of alcoholic extract when applied concomitantly with the bacteria. This result was similar of Bordeaux mixture, indicating a control by direct antimicrobial activity. There was no systemic resistance induction for all the treatments. The peroxidases induction was due to infectious pathogen process and not to the treatments with alcoholic extract [37] Other examples Below are presented examples in which various plants were tested together. Extracts of Cymbopogon citratus, Curcuma longa and Rosmarinus officinalis were used to control late season diseases (Septoria glycines and Cercospora kikuchii) and powdery mildew (Microsphaera diffusa) of soybean in field conditions. The treatment with curcumin at 50 mg/l provided total weight of soybean seeds 29% bigger than the 1037

6 A. Méndez-Vilas (Ed.) obtained with the standard treatment with the fungicide pyraclostrobin + epoxiconazole (0.6 L/ha). Extracts of C. citratus and C. longa a 5% provided the same level of control of late season diseases that the fungicide. C. citratus and R. officinalis a 5% and C. longa at 5 and 10% had the same efficiency of the fungicide to control powdery mildew. Some extracts, as C. longa at 10%, besides efficient control of late season diseases and powdery mildew in soybean, resulted in a productivity 16% bigger (equivalent to 535 kg/ha) in relation to treatment with fungicide [38]. Experiments in field conditions (organic growth) with two strawberry cultivars, Camarosa and Dover, were done to verify the control of Rhizopus sp. and Colletotrichum fragariae, using aqueous extracts of rosemary and rue (Ruta graveolens). It was verified to Camarosa cultivar that the treatments with rosemary reduced 51% the disease incidence when compared to the control treatment (water). These values were similar to the treatment with Bordeaux mixture (standard treatment used by farmers in organic growth), in which the reduction of incidence was of 52%. For the treatment with rue, the reduction of disease was of 39%. These results reflected directly in production of strawberries, where the treatments with rosemary increase the production in 214%, comparing to control treatment, and in 58% in relation to treatment with Bordeaux mixture. For rue, the increase of fruits production was of 223% in relation to the control treatment, and 67% when comparing with Bordeaux mixture. In the case of extract of rosemary and rue at 10%, the increment in production was around 326%. The treatment with Bordeaux mixture increased in 156% the production in relation to the control treatment. To Dover cultivar, the treatments with rosemary increase 127% the strawberries production. For rue, the increase was 131%, while the treatment with Bordeaux mixture increased in 154% the fruit production [39]. It were evaluated, in vitro, the fungitoxicity of aqueous extracts of Achillea millefolium, Artemisia camphorata, Cymbopogon citratus and Rosmarinus officinalis against Cladosporium fulvum, and the protective effect of these extracts in tomato plants under greenhouse conditions. To evaluate antifungal activity, extracts were incorporated to PDA (potato-dextrose-agar), with observation of inhibition of mycelial growth, sporulation and spores germination. The plant protection was verified by preventive spraying, 72 hours before inoculation, of aqueous extracts, in 10% and 20% concentration, in the first pair of leaves. The severity was evaluated 20 days after inoculation, which occurred in both the first and second pair of leaves. The activity of the plant-defense enzyme peroxidase was evaluated in leaves 72 hours after treatments. It was verified that extracts inhibit the mycelial growth and had significant reduction in sporulation and spore germination, mainly by extracts of A. camphorata and R. officinalis, in concentration of 20% and 40%, which reduced the spore production 85.72% and 93.49%, respectively. In relation to the in vivo treatments there was observed a reduction in the number of lesions, with a possible resistance induction, mainly to A. camphorata and R. officinalis extracts. The peroxidase activity had no significant increment in relation to the control treatment [40]. The hydrolates of Helietta apiculata (HA), Conyza canadensis (CC) and Cymbopogon nardus (CN) were used in the concentrations of 1, 5, 10, 15, 20 and 25%. In the phytoalexins assay, sorghum etiolated mesocotyls were used. The antibacterial effect was evaluated on the growth of the bacterium Xanthomonas campestris pv. campestris in liquid nutrient medium and antibiotic (oxytetracycline 22.5 mg/l + streptomycin 225 mg/l) was used as control treatment. The antifungal effect was evaluated on the mycelial growth, sporulation, conidia germination and development of germtubes of Alternaria brassicae. The fungicide azoxystrobin (0.08 g a.i./l) was used as control treatment. There was increment in the phytoalexins synthesis with the increase in hydrolates concentration. The better result was obtained with CN that promoted increase 4.3 times larger in relation to the value of the control treatment with water, followed by HA (2.5 times) and CC (2.1 times). Similar results were obtained to the antibacterial activity with the following inhibition results (%) in the development of the bacterium: CN: 29.8, HA: 14.9, CC: 14.6 and antibiotic: The main antifungal effect was observed on the development of the germ-tubes. Hydrolates of CC and HA showed inhibition of up to 69.2 and 56.2%, respectively, being similar to the fungicide azoxystrobin. The hydrolate of CN did not show antifungal effect. These results indicate the presence of phytoalexins elicitors, and antibacterial and antifungal compounds in those hydrolates, however in low concentrations [41]. Two experiments were conducted to evaluate the antimicrobial activity of alternative products against Colletotrichum musae. First, bananas naturally infected by anthracnose were immersed into Azadirachta indica and citric extracts at 2 and 4% (v/v) for 3 minutes and stored for 11 days under environmental conditions. Next, other the fruits were immersed into essential oil emulsions of Allium sativum, Copaifera langsdorfii, Cinnamomum zeylanicum and Eugenia caryophyllata at 5% for 3 minutes but stored for 13 days. Fruits immersed into distilled water were used as controltreatments. One hundred percent of disease incidence observed in the control-treatment was similar to 88.89% observed in the extract of A. indica at 2%. The control-treatment presented disease severity of 75.13% and percentage of disease control of 20.85%. Fruits immersed into distilled water were significantly different from those immersed into citric extracts which had the highest effectiveness. Citric extract at 4% was the most efficient treatment because the disease incidence was 19.44%, the disease severity was 9.34% and the disease control was 90.16%. Less severity and, consequently, more disease control were achieved by immersing the fruits into the essential oil of A. sativum or treatments with C. langsdorfii, E. caryophyllata and C. zeylanicum [42]. The fungitoxicity of crude extracts and essential oils of Achillea millefolium, Cymbopogon citratus, Eucalyptus citriodora and Ageratum conyzoides on the fungus Didymella bryoniae was verified in vitro by means of germination of spores and mycelial growth. In addition, some observations were made using scanning electron microscopy (SEM) to detect possible alterations on the hyphae of Didymella bryoniae. The results revealed that crude extracts of E. citriodora 1038

7 Science against microbial pathogens: communicating current research and technological advances and A. conyzoides were more effective in inhibiting the mycelial growth of D. bryoniae whereas in the germination of spores A. conyzoides and A. millefolium were responsible for most of the inhibition, namely, 52 and 46%, respectively. The essential oils of C. citratus, A. conyzoides and E. citriodora provided 100% inhibition of the mycelial growth and germination of spores of D. bryoniae. SEM observations revealed alterations in the growth pattern of hyphae of D. bryoniae when the essential oil of A. millefolium was present [43]. 3. Fungal extracts 3.1 Pycnoporus sanguineus Aqueous extracts of Pycnoporus sanguineus, obtained from liquid medium culture filtrate, was used to verify in vitro antimicrobial activity against Pseudocercospora griseola and to induced resistance in bean (Phaseolus vulgaris) against angular leaf spot. The role of the 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 had been constituted by extracts from liquid medium culture filtrate of P. sanguineus. Water, acibenzolar-s-methyl (ASM: 75 mg i.a./l) and fungicide (azoxystrobin: 40 mg i.a./l) were the control treatments. In vitro, the extract of liquid medium culture filtrate did not inhibit the mycelial growth, sporulation and conidia germination of P. griseola. Severity was reduced in 82% and 49% 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 extracts. These results indicate the potential of P. sanguineus liquid medium culture filtrate for alternative control of the angular leaf spot in bean plants [5]. In another assay, aqueous extract of Pycnoporus sanguineus mycelium was used to verify the in vitro antimicrobial activity against Pseudocercospora griseola and to induce resistance in bean against this pathogen. The role of the 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 had been constituted by extracts from mycelium of P. sanguineus and water, acibenzolar-s-methyl (ASM: 75 mg/l) and fungicide (azoxystrobin: 40 mg/l) were the control treatments. In vitro, the extract of mycelium did not inhibit the mycelial growth, sporulation and conidia germination of P. griseola. 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 extracts. These results indicate the potential of P. sanguineus for alternative control of the angular leaf spot in bean plants [6]. To in vitro assays against Xanthomonas axonopodis pv. phaseoli were used aqueous extracts from basidiocarp, mycelium and culture filtrate of Pycnoporus sanguineus in concentrations of 1, 5, 10, 15 and 20%, with water, acibenzolar-s-methyl (ASM mg a.i./l) and antibiotic (oxytetracycline 22.5 mg/l + streptomycin 225 mg/l) as control treatments. For in vivo assays against common bacterial blight in bean were evaluated disease severity and the activities of peroxidase, polyphenol oxidase, β-1.3 glucanase and phenylalanine ammonia-lyase, using extracts of mycelium, basidiocarp and culture filtrate of P. sanguineus at 5% and 10%. It was verified antibacterial activity only for culture filtrate in concentrations up 15% and for all concentrations of basidiocarp extract. The results of in vivo assays indicated the potential of basidiocarps extracts from P. sanguineus for the control of Xanthomonas axonopodis pv. phaseoli in bean, which can occur either by direct antimicrobial activity and resistance induction involving the activation of those plant defense enzymes [7]. Dichloromethane, hexane and ethanol extracts obtained from Pycnoporus sanguineus basidiocarp, in concentrations of 100, 250, 500 and 750 mg/l were used for phytoalexins induction and peroxidases activity in soybean cotyledons and sorghum etiolated mesocotyls. The acybenzolar-s-methyl (ASM) (200 mg/l of commercial product) and distilled water + Tween 20 (0.5%) were used as positive and negative control treatments, respectively. For the phytoalexins assay in sorghum mesocotyls, the hexane-extract in concentration of 750 mg/l provided the highest induction, however without differing significantly from ASM. For the phytoalexins assay in the soybean cotyledons, the P. sanguineus extracts did not induce significantly different in relation to positive and negative control treatments, and showed a suppression of the glyceollin synthesis for the dichloromethane-extract. In relation to peroxidases, the dichloromethaneextracts for sorghum and soybean and ethanol-extract for soybean had inhibited the enzymatic activity. The induction verified for the hexane-extract in sorghum was not different from ASM control. The specific activity of peroxidase in soybean was inhibited by the ethanol-extract and it was induced by the hexane, but with no differences of the treatment with ASM [44]. In another experiment, the in situ detection of reactive oxygen species (ROS) was searched in bean plants treated with aqueous extracts of the mycelium (AEM) and basidiocarps (AEB) from Pycnoporus sanguineus and inoculated after three days with Colletotrichum lindemuthianum. It was evaluated ROS formation, mainly hydrogen peroxide (H2O2) and superoxide (O2.-), at 48, 96 and 192 hours after the inoculation (hai). Distillated water, azoxystrobin (40 mg a.i./l) and acibenzolar-s-methyl (75 mg a.i./l) were the control treatments. It was possible to detect H2O2 at 48 hai only to the treatment with basidiocarp extract. The O2.- was detected mainly to the treatment with mycelium extract at 48 hai. All the treatments showed reaction for H2O2 and O2.- in epidermal and mesophyllic cells at 192 hai, probably due the 1039

8 Science against microbial pathogens: communicating current research and technological advances infection development. These results showed the potential of P. sanguineus extracts for the ROS s induction in bean plants in early infection process, which can reduce the anthracnose severity [45]. 3.2 Lentinula edodes and Agaricus blazei With the purpose of finding alternative ways to control Bipolaris sorokiniana and Puccinia recondita f.sp. tritici, the in vitro fungitoxic effect of L. edodes and A. blazei mushrooms was tested on these fungi. The aqueous extract of both mushrooms did not have significant effect on mycelial growth and spore germination of B. sorokiniana. On the other hand, the mushroom extracts inhibited the germination of P. recondita f.sp. tritici uredospores, particularly on the L. edodes, which exhibited the largest inhibition of spore germination (52%) [4]. 3.3 Saccharomyces boulardii Saccharomyces yeast compounds are been used as resistance elicitor or for biological control in many pathosystems. Thus, the aim of this research was to verify the phytoalexins induction in sorghum mesocotyls and soybean cotyledons by using Saccharomyces boulardii commercial product (Floratil - MERCK) (2x106 cells/mg) and yeast cell mass obtained from liquid culture in YEPG medium (with 7, 14, 21, 28 and 35 days old), both at concentrations of 0.005, 0.05, 0.5, 5, 15 and 25 mg/ml and its culture filtrate in concentrations of 0.01, 0.1, 1, 5, 10 and 20%. As control treatments were used distillated water and Saccharomyces cerevisiae (25 mg of commercial product/ml) to soybean essays and distillated water and acibenzolar-s-methyl (125 mg of active ingredient/l) to sorghum essays. To soybean the three tested products from S. boulardii showed dose-dependent effect with R2 of 0.64, 0.94 and 0.98 to culture filtrate, cell suspension and commercial product of S. boulardii, respectively, with no effect of culture time of yeasts on phytoalexin induction. To sorghum phytoalexins induction, just culture filtrate and Floratil had dose-dependent effect with R2 of 0.63 and 0.94, respectively, and cell suspension of S. boulardii showed dependence of culture time with R2 of Thus, S. boulardii and its derivates induce phytoalexins and have potential to be used as elicitor in induction resistance assays on pathosystems involving sorghum and soybean plants [12]. Acknowledgements: The authors express their acknowledgement to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for research grants and fellowships (JRS and KRFSE), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Financiadora de Estudos e Projetos (FINEP) and Fundação Araucária to carry out these researches. References [1] Stangarlin JR, Schwan-Estrada KRF, Cruz MES, Nozaki MH. Medicinal plants and alternative control of phytopathogens. Biotecnologia Ciência & Desenvolvimento. 1999;11: [2] Schwan-Estrada KRF, Stangarlin JR. Extracts and essential oils of medicinal plants in the resistance induction against plant pathogens. In: Cavalcanti LS, Di Piero RM, Cia P, Pscholati SF, Resende MLV, Romeiro RS, eds. Resistance induction in plants against pathogens and insects. Piracicaba: FEALQ, 2005: [3] Di Piero RM, Novaes QS, Pascholati SF. 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UNIOESTE: Universidade Estadual do Oeste do Paraná [40] Itako AT, Schwan-Estrada KRF, Stangarlin JR, Tolentino Júnior JB, Cruz MES. Control of Cladosporium fulvum in tomato plants by extracts of medicinal plants. Arquivos do Instituto Biológico. 2009;76: [41] Franzener G, Martinez-Franzener AS, Stangarlin JR, Czepak MP, Schwan-Estrada KRF, Cruz MES. Antibacterial, antifungal and phytoalexins induction activities of hydrolates of medicinal plants. Semina Ciências Agrárias. 2007;28: [42] Cruz MES. Alternative products on the control of post-harvest diseases in banana (Musa paradisiaca L.), apple (Malus domestica Borkh) and orange (Citrus sinensis (L.) Osbeck). (PhD Thesis). UEM: Universidade Estadual Maringá;