An account of Colletotrichum species associated with anthracnose of Atractylodes ovata in South Korea based on morphology and molecular data.

Ovate-leaf atractylodes (OLA) (Atractylodes ovata) is a well-known medicinal plant in Korea; its dried rhizome and root extracts are used in herbal medicine. However, anthracnose is a great challenge to the OLA cultivation in South Korea. Colletotrichum spp. is a major group of plant pathogens responsible for anthracnose on a range of economically important hosts. Its occurrence on OLA remains unresolved. To investigate the diversity, morphology, phylogeny, and biology of Colletotrichum spp., 32 fungal isolates were obtained from 30 OLA-affected leaves collected from five different farms, in two regions in South Korea, Mungyeong and Sangju. The phylogenetic analysis with four or five gene loci (ITS, TUB2, ACT, GAPDH, and CHS-1) along with morphology of 26 representative isolates delineated six previously known Colletotrichum species including C. fructicola, C. gloeosporioides sensu stricto (s.s), C. cigarro, C. plurivorum, C. siamense and C. sojae, and one new species, described here as C. ovataense. Amongst these species, C. gloeosporioides s.s. and C. plurivorum were the most prevalent species. A pathogenicity test on the detached leaves revealed that different Colletotrichum species presented a distinct degree of virulence, confirming Koch's postulates. In this study, C. fructicola, C. cigarro, C. plurivorum, C. siamense, and C. sojae were reported from A. ovata for the first time, as the causal agent of ovate-leaf atractylodes anthracnose. Understanding the diversity and biology of the Colletotrichum species population will help in managing this disease.

Effects of Bacillus Subtilis CF-3 VOCs Combined with Heat Treatment on the Control of Monilinia fructicola in Peaches and Colletotrichum gloeosporioides in Litchi Fruit.

In order to study the effect of volatile organic compounds (VOCs) produced by Bacillus subtilis CF-3 combined with heat treatment on Monilinia fructicola in peach and Colletotrichum gloeosporioides in litchi fruit, fruits were treated with B. subtilis CF-3 VOCs and hot air alone or in combination. The quality indexes of peach and litchi fruit after treatment and the changes in defense-related enzymes were measured. The results showed that the B. subtilis CF-3 VOCs combined with heat treatment could significantly reduce the rot index of peach and litchi fruit, and effectively maintain firmness and soluble solids content, as well as reduce weight loss of fruits. The combined treatment effectively enhanced the activity of peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT), and superoxide dismutase (SOD) than either treatment alone, and enhanced the resistance of fruit to pathogenic fungi by activating disease-resistant enzymes (phenylalanine ammonia-lyase [PAL], chitinase [CHI], ß-1, 3-glucanase [GLU]) activity. In this study, B. subtilis CF-3 VOCs combined with heat treatment maintained the quality and delayed the decline of peach and litchi fruit, providing a theoretical basis for future applications. PRACTICAL APPLICATION: The combination of B. subtilis CF-3 VOCs and heat treatment reduce the extent of M. fructicola and C. gloeosporioides. The combination maintain the quality of peach and litchi better. The combination obviously improve the activity of defense-related enzyme in fruit.

Colletotrichum Species Associated with Japanese Plum (Prunus salicina) Anthracnose in South Korea.

A total of 24 Colletotrichum isolates were isolated from diseased Japanese plum (Prunus salicina) fruits showing chlorotic regions with whitish-brown sunken necrotic lesions and phylogenetic relationships among the collected Colletotrichum isolates were determined. A subset of 11 isolates was chosen for further taxonomic study based on morphology and molecular characteristics identified using the internal transcribed spacer (ITS) and beta-tubulin (TUB2) genes. Isolates in the C. acutatum complex were analyzed using partial sequencing of five gene regions (ITS, GAPDH, ACT, TUB2, and CHS), and C. gloeosporioides sensu lato (s.l.) isolates were analyzed using seven gene regions (ITS, TUB2, GAPDH, ACT, CAL, CHS-1, and ApMat). Morphological assessments in combination with phylogenetic analysis delineated four species of Colletotrichum including C. gloeosporioides sensu stricto (s.s.), C. nymphaeae, C. foriniae, and C. siamense; these data identify Colletotrichum fioriniae and C. siamense two new species associated with plum anthracnose in South Korea. Finally, the pathogenicity of these four species in the development of plum anthracnose in South Korea was confirmed by inoculations of plum fruit.

Alternative control of grape rots by essential oils of two Eucalyptus species.

BACKGROUND: Essential oils (EOs) are volatile natural compounds produced by plant secondary metabolism, and some of them exhibit antimicrobial activity. The objective of the present study was to determine the chemical composition the EOs of Eucalyptus staigeriana and Eucalyptus globulus, and their effect in vitro and in vivo against Botrytis cinerea and Colletotrichum acutatum, the most important fungal rot diseases of grapes. Moreover, grapes collected from field experiments were used to evaluate the impact of the alternative control on the alcoholic fermentation and wine composition. RESULTS: The major compound of E. staigeriana EO were citral 30.91% (19.74% geranial, 11.17% neral), 1.8-cineole (24.59%) and limonene (19.47%), while 1.8-cineole represented 68.26% of E. globulus EO. The two EOs showed in vitro antifungal activity against both pathogens. Eucalyptus staigeriana EO exhibited the highest activity inhibiting mycelial growth (MG) and conidial germination at 0.5 µL mL-1 . Moreover, this EO was able to reduce the incidence and severity of grey rot caused by B. cinerea and the severity of ripe rot caused by C. acutatum The alternative control did not significantly influence alcoholic fermentation, the physicochemical characteristics, and the volatile composition of wines. CONCLUSION: These results are promising and indicate that E. staigeriana EO might be further investigated as a natural alternative for the control of fungal rots on wine grapes. © 2019 Society of Chemical Industry.

Bacterial communities associated with anthracnose symptomatic and asymptomatic leaves of guarana, an endogenous tropical crop, and their pathogen antagonistic effects.

Plants are colonized by diverse microorganisms that can substantially impact their health and growth. Understanding bacterial diversity and the relationships between bacteria and phytopathogens may be key to finding effective biocontrol agents. We evaluated the bacterial community associated with anthracnose symptomatic and asymptomatic leaves of guarana, a typical tropical crop. Bacterial communities were assessed through culture-independent techniques based on extensive 16S rRNA sequencing, and cultured bacterial strains were evaluated for their ability to inhibit the growth of Colletotrichum sp. as well as for enzyme and siderophore production. The culture-independent method revealed that Proteobacteria was the most abundant phylum, but many sequences were unclassified. The emergence of anthracnose disease did not significantly affect the bacterial community, but the abundance of the genera Acinetobacter, Pseudomonas and Klebsiella were significantly higher in the symptomatic leaves. In vitro growth of Colletotrichum sp. was inhibited by 11.38% of the cultured bacterial strains, and bacteria with the highest inhibition rates were isolated from symptomatic leaves, while asymptomatic leaves hosted significantly more bacteria that produced amylase and polygalacturonase. The bacterial isolate Bacillus sp. EpD2-5 demonstrated the highest inhibition rate against Colletotrichum sp., whereas the isolates EpD2-12 and FD5-12 from the same genus also had high inhibition rates. These isolates were also able to produce several hydrolytic enzymes and siderophores, indicating that they may be good candidates for the biocontrol of anthracnose. Our work demonstrated the importance of using a polyphasic approach to study microbial communities from plant diseases, and future work should focus on elucidating the roles of culture-independent bacterial communities in guarana anthracnose disease.

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts.

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral extraction methods and coverslip bioassays were developed utilizing Colletotrichum fioriniae. In blueberry and cranberry, this pathogen is optimally controlled by applying fungicides during the bloom period because of the role flowers play in the initial stages of infection. The protocol detailed here describes how floral extracts (FE) were obtained using water-, chloroform-, and field rainwater-based methods for later use in corresponding glass coverslip bioassays. Each FE served to provide a different set of information: response of C. fioriniae to mobilized floral chemical cues in water (water-based), pathogen response to flower and fruit surface waxes (chloroform-based), and field-based monitoring of collected floral rainwater, moving in vitro observations to an agricultural setting. The FE is broadly described as either water- or chloroform-based, with an appropriate bioassay described to compensate for the inherent differences between these two materials. Rainwater that had run off flowers was collected in unique devices for each crop, alluding to the flexibility and application of this approach for other crop systems. The bioassays are quick, inexpensive, simple, and provide the ability to generate spatiotemporal and site-specific information about the presence of stimulatory floral compounds from various sources. This information will ultimately better inform disease management strategies, as FE decrease the time needed for infection to occur, thus providing insight into changing risks for pathogen infection over the growing season.

ZnO-based nanofungicides: Synthesis, characterization and their effect on the coffee fungi Mycena citricolor and Colletotrichum sp.

In this work we compare the antifungal capacity of zinc oxide nanoparticles (ZnO-NPs) synthesized by a chemical route and a ZnO-based nanobiohybrid obtained by green synthesis in an extract of garlic (Allium sativum). To find out the characteristics of the materials synthesized, X-ray diffraction (XRD), IR spectroscopy and absorption in UV-Vis were used, as well as both scanning (SEM) and transmission (TEM) electron microscopy. The results showed that the samples obtained were of nanometric size (<100 nm), with a predominance of the wurtzite crystal phase of ZnO and little crystallization of the nanobiohybrids. Their antifungal capacity on two pathogenic fungi of coffee, Mycena citricolor (Berk and Curt) and Colletotrichum sp. was also evaluated. Both nanomaterials showed an efficient antifungal capacity, particularly the nanobiohybrids, with ~97% inhibition in growth of M. citricolor, and ~93% for Colletotrichum sp. The microstructural study with high resolution optical (HROM) and ultra-structural microscopy (using TEM) carried out on the fungi treated with the synthesized nanomaterials showed a strong nanofungicidal effect on the vegetative and reproductive structures and fungal cell wall, respectively. The inhibition of the growth of the fungi and micro and ultra-structural affectations were explained considering that the size of the nanomaterials allows them to pass easily through the cell membranes. This indicates that they can be absorbed easily by the fungi tested here, causing cellular dysfunction. Nanofungicide effects are also attributable to the unique properties of nanomaterials, such as the high surface-to-bulk ratio of atoms and their surface physicochemical characteristics that could directly or indirectly produce reactive oxygen species (ROS), which affect the proteins of the cell wall.

Effects of Phenolic Compounds on Growth of Colletotrichum spp. In Vitro.

Colletotrichum acutatum is responsible for anthracnose fruit rot, one of the most devastating diseases in strawberry. Phenolic compounds have been described as contributors to anthracnose resistance in strawberry (Fragaria x ananassa, Duch.). Six isolates of Colletotrichum acutatum and four isolates of three other Colletotrichum species, C. gloeosporioides, C. fragariae, and C. graminicola, associated with disease symptoms were investigated in this study. The potential inhibitory effect of phenolic acids (gallic acid, caffeic acid, chlorogenic acid, ferulic acid, trans-cinnamic acid, p-coumaric acid, salicylic acid), flavonoids (catechin, quercetin, naringenin), and ellagic acid, which are naturally found in strawberry, were screened against two different spore suspension concentrations of the Colletotrichum isolates at 5, 10, 50 mM in vitro. Among the phenolic acids and flavonoids tested in this study, only trans-cinnamic acid, ferulic acid, and p-coumaric acid inhibited fungal growth. The inhibitory effects were concentration-dependent but also varied with the spore suspension concentration of the isolates. The results demonstrated that trans-cinnamic acid had the greatest inhibitory effect on all Colletotrichum spp. isolates tested.

Evaluation of a Pomegranate Peel Extract as an Alternative Means to Control Olive Anthracnose.

Olive anthracnose is caused by different species of Colletotrichum spp. and may be regarded as the most damaging disease of olive fruit worldwide, greatly affecting quality and quantity of the productions. A pomegranate peel extract (PGE) proved very effective in controlling the disease. The extract had a strong in vitro fungicidal activity against Colletotrichum acutatum sensu stricto, was very effective in both preventive and curative trials with artificially inoculated fruit, and induced resistance in treated olive tissues. In field trials, PGE was significantly more effective than copper, which is traditionally used to control the disease. The highest level of protection was achieved by applying the extract in the early ascending phase of the disease outbreaks because natural rots were completely inhibited with PGE at 12 g/liter and were reduced by 98.6 and by 93.0% on plants treated with PGE at 6 and 3 g/liter, respectively. Two treatments carried out 30 and 15 days before the expected epidemic outbreak reduced the incidence of the disease by 77.6, 57.0, and 51.8%, depending on the PGE concentration. The analysis of epiphytic populations showed a strong antimicrobial activity of PGE, which sharply reduced both fungal and bacterial populations. Because PGE was obtained from a natural matrix using safe chemicals and did not have any apparent phytotoxic effect on treated olive fruit, it may be regarded as a safe and effective natural antifungal preparation to control olive anthracnose and improve olive productions.

Synergistic mixtures of chitosan and Mentha piperita L. essential oil to inhibit Colletotrichum species and anthracnose development in mango cultivar Tommy Atkins.

This study assessed the efficacy of chitosan (CHI) and Mentha piperita L. essential oil (MPEO) alone or in combination to control the mycelial growth of five different Colletotrichum species, C. asianum, C. dianesei, C. fructicola, C. tropicale and C. karstii, identified as potential anthracnose-causing agents in mango (Mangifera indica L.). The efficacy of coatings of CHI and MPEO mixtures in controlling the development of anthracnose in mango cultivar Tommy Atkins was evaluated. CHI (2.5, 5, 7.5 and 10 mg/mL) and MPEO (0.3, 0.6, 1.25, 2.5 and 5 µL/mL) alone effectively inhibited mycelial growth of all tested Colletotrichum strains in synthetic media. Mixtures of CHI (5 or 7.5 mg/mL) and MPEO (0.3, 0.6 or 1.25 µL/mL) strongly inhibited mycelial growth and showed additive or synergistic inhibitory effects on the tested Colletotrichum strains based on the Abbott index. The application of coatings of CHI (5 or 7.5 mg/mL) and MPEO (0.6 or 1.25 µL/mL) mixtures that presented synergistic interactions decreased anthracnose lesion severity in mango artificially contaminated with either of the tested Colletotrichum strains over 15 days of storage at 25 °C. The anthracnose lesion severity in mango coated with the mixtures of CHI and MPEO was similar or lower than those observed in mango treated with the synthetic fungicides thiophanate-methyl (10 µg a.i./mL) and difenoconazole (0.5 µg a.i./mL). The application of coatings containing low doses of CHI and MPEO may be an effective alternative for controlling the postharvest development of anthracnose in mango cultivar Tommy Atkins.

Application and bioactive properties of CaTI, a trypsin inhibitor from Capsicum annuum seeds: membrane permeabilization, oxidative stress and intracellular target in phytopathogenic fungi cells.

BACKGROUND: During the last few years, a growing number of antimicrobial peptides have been isolated from plants and particularly from seeds. Recent results from our laboratory have shown the purification of a new trypsin inhibitor, named CaTI, from chilli pepper (Capsicum annuum L.) seeds. This study aims to evaluate the antifungal activity and mechanism of action of CaTI on phytopathogenic fungi and detect the presence of protease inhibitors in other species of this genus. RESULTS: Our results show that CaTI can inhibit the growth of the phytopathogenic fungi Colletotrichum gloeosporioides and C. lindemuthianum. CaTI can also permeabilize the membrane of all tested fungi. When testing the inhibitor on its ability to induce reactive oxygen species, an induction of reactive oxygen species (ROS) and nitric oxide (NO) particularly in Fusarium species was observed. Using CaTI coupled to fluorescein isothiocyanate (FITC), it was possible to determine the presence of the inhibitor inside the hyphae of the Fusarium oxysporum fungus. The search for protease inhibitors in other Capsicum species revealed their presence in all tested species. CONCLUSION: This paper shows the antifungal activity of protease inhibitors such as CaTI against phytopathogenic fungi. Antimicrobial peptides, among which the trypsin protease inhibitor family stands out, are present in different species of the genus Capsicum and are part of the chemical arsenal that plants use to defend themselves against pathogens. © 2017 Society of Chemical Industry.

Cation-Stress-Responsive Transcription Factors SltA and CrzA Regulate Morphogenetic Processes and Pathogenicity of Colletotrichum gloeosporioides.

Growth of Colletotrichum gloeosporioides in the presence of cation salts NaCl and KCl inhibited fungal growth and anthracnose symptom of colonization. Previous reports indicate that adaptation of Aspergillus nidulans to salt- and osmotic-stress conditions revealed the role of zinc-finger transcription factors SltA and CrzA in cation homeostasis. Homologs of A. nidulans SltA and CrzA were identified in C. gloeosporioides. The C. gloeosporioides CrzA homolog is a 682-amino acid protein, which contains a C2H2 zinc finger DNA-binding domain that is highly conserved among CrzA proteins from yeast and filamentous fungi. The C. gloeosporioides SltA homolog encodes a 775-amino acid protein with strong similarity to A. nidulans SltA and Trichoderma reesei ACE1, and highest conservation in the three zinc-finger regions with almost no changes compared to ACE1 sequences. Knockout of C. gloeosporioides crzA (ΔcrzA) resulted in a phenotype with inhibited growth, sporulation, germination and appressorium formation, indicating the importance of this calciu006D-activated transcription factor in regulating these morphogenetic processes. In contrast, knockout of C. gloeosporioides sltA (ΔsltA) mainly inhibited appressorium formation. Both mutants had reduced pathogenicity on mango and avocado fruit. Inhibition of the different morphogenetic stages in the ΔcrzA mutant was accompanied by drastic inhibition of chitin synthase A and B and glucan synthase, which was partially restored with Ca2+ supplementation. Inhibition of appressorium formation in ΔsltA mutants was accompanied by downregulation of the MAP kinase pmk1 and carnitine acetyl transferase (cat1), genes involved in appressorium formation and colonization, which was restored by Ca2+ supplementation. Furthermore, exposure of C. gloeosporioides ΔcrzA or ΔsltA mutants to cations such as Na+, K+ and Li+ at concentrations that the wild type C. gloeosporioides is not affected had further adverse morphogenetic effects on C. gloeosporioides which were partially or fully restored by Ca2+. Overall results suggest that both genes modulating alkali cation homeostasis have significant morphogenetic effects that reduce C. gloeosporioides colonization.

Metabolic Changes of Caffeine in Tea Plant (Camellia sinensis (L.) O. Kuntze) as Defense Response to Colletotrichum fructicola.

Tea plant (Camellia sinensis) is one of the most economically valuable crops in the world. Anthracnose can affect the growth of leaves and cause serious yield losses of tea. Tea plants are rich in secondary metabolites; however, their roles in resistance to anthracnose are unclear. Herein we compared the contents of total phenolics, catechins, and caffeine in two cultivars with different resistances to anthracnose during Colletotrichum fructicola infection. (-)-Epigallocatechin-3-gallate (EGCG), (+)-catechin (C), caffeine, and critical regulatory genes were induced in C. fructicola-resistant tissues. In vitro antifungal tests showed that caffeine more strongly inhibited mycelial growth than tea polyphenols and catechins. Both electron microscopy and bioactivity analysis results showed that caffeine can affect mycelial cell walls and plasma membranes. Through promoter sequences analysis, a number of stress response-related cis-acting elements were identified in S-adenosylmethionine synthetase and tea caffeine synthase. These results demonstrated that (-)-EGCG, (+)-C, and caffeine may be involved in the resistance of tea plants to anthracnose.

Endophytic cultivable bacterial community obtained from the Paullinia cupana seed in Amazonas and Bahia regions and its antagonistic effects against Colletotrichum gloeosporioides.

Guarana (Paullinia cupana var. sorbilis) is a plant from the Amazonas region with socio-economic importance. However, guarana production has been increasingly affected by unfavorable conditions resulting from anthracnose, caused by the Colletotrichum fungal genus, which primarily affects mainly the Amazonas region. The aim of the present study was to isolate bacterial endophytes from the seeds of guarana plants obtained from Amazonas region and the Northeast state of Bahia, a region where this disease is not a problem for guarana plantations. The number of bacterial Colony Forming Units (CFU/g seeds) was 2.4 × 10(4) from the Bahia and 2.9 × 10(4) from the Amazonas region. One hundred and two isolated bacteria were evaluated in vitro against the phytopathogenic strain Colletotrichum gloeosporioides L1. These isolates were also analyzed for the enzymatic production of amylase, cellulase, protease, pectinase, lipase and esterase. Approximately 15% of isolates, showing high antagonistic activity, and the production of at least one enzyme were identified through the partial sequencing of 16S rDNA. The genus Bacillus was the most frequently observed, followed by Paenibacillus, Ochrobactrum, Microbacterium and Stenotrophomonas. Proteolytic activity was observed in 24 isolates followed by amylolytic, pectinolytic and cellulolytic activities. No esterase and lipase production was detected. Most of the isolates, showing antagonistic effects against C. gloeosporioides and high enzymatic activities, were isolated from the anthracnose-affected region. A biocontrol method using the endophytes from guarana seeds could be applied in the future, as these bacteria are vertically transferred to guarana seedlings.

Application of lemongrass oil in vapour phase for the effective control of anthracnose of 'Sekaki' papaya.

AIMS: To evaluate the potential use of lemongrass essential oil vapour as an alternative for synthetic fungicides in controlling anthracnose of papaya. METHODS AND RESULTS: Lemongrass oil used in the study was characterized using gas chromatography-flame ionization detection (GC-FID) before it was tested against anthracnose of papaya in vitro and in vivo. The GC-FID analysis showed that geranial (45·6%) and neral (34·3%) were the major components in lemongrass oil. In vitro study revealed that lemongrass oil vapour at all concentrations tested (33, 66, 132, 264 and 528 µl l(-1) ) suppressed the mycelial growth and conidial germination of Colletotrichum gloeosporioides. For the in vivo study, 'Sekaki' papaya were exposed to lemongrass oil fumigation (0, 7, 14, 28 µl l(-1) ) for 18 h and at room temperature for 9 days. Lemongrass oil vapour at the concentration of 28 µl l(-1) was most effective against anthracnose of artificially inoculated papaya fruit while quality parameters of papaya were not significantly altered. CONCLUSIONS: This suggests that lemongrass oil vapour can control anthracnose disease development on papaya without affecting its natural ripening process. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential practical application of this technology can reduce reliance on synthetic fungicides for the control of postharvest diseases in papaya.

Antifungal activity of compounds extracted from Cortex Pseudolaricis against Colletotrichum gloeosporioides.

Cortex Pseudolaricis is the root bark of Pseudolarix amabilis Rehder, found only in China, and has been widely used in folk antifungal remedies in traditional Chinese medicine. In order to find the natural antifungal agents against mango anthracnose, eight compounds, namely pseudolaric acid A (1), ethyl pseudolaric acid B (2), pseudolaric acid B (3), pseudolaric acid B-O-ß-d-glucoside (4), piperonylic acid (5), propionic acid (6), 3-hydroxy-4-methoxybenzoic acid (7), and 4-(3-formyl-5-methoxyphenyl) butanoic acid (8) were isolated from the ethanol extracts of Cortex Pseudolaricis by bioassay-guided fractionation and evaluated for in vitro antifungal activity against Colletotrichum gloeosporioides Penz. Results demonstrated that all of the eight compounds inhibited the mycelial growth of C. gloeosporioides at 5 µg/mL. Among them, pseudolaric acid B and pseudolaric acid A showed the strongest inhibition with the EC50 values of 1.07 and 1.62 µg/mL, respectively. Accordingly, both Pseudolaric acid B and Pseudolaric acid A highly inhibited spore germination and germ tube elongation of C. gloeosporioides. Dipping 100 µg/mL pseudolaric acid B treatment exhibited more effective suppression on postharvest anthracnose in mango fruit when compared to the same concentration of carbendazim. Scanning electron microscopy observations revealed that pseudolaric acid B caused alterations in the hyphal morphology of C. gloeosporioides, including distortion, swelling, and collapse. Pseudolaric acid B caused the mycelial apexes to show an abnormal growth in dimensions with multiple ramifications in subapical expanded areas with irregular shape. These findings warrant further investigation into optimization of pseudolaric acid B to explore a potential antifungal agent for crop protection.

Influência de diferentes extratos aquosos de plantas medicinais no desenvolvimento de Colletotrichum gloeosporioides e de Fusarium moniliforme / Influence of different aqueous extracts of medicinal plants in the development of Colletotrichum gloeosporioides and Fusarium moniliforme

O objetivo deste trabalho foi avaliar a influência dos extratos aquosos das plantas medicinais alecrim, alho, cravo-da-índia, sálvia, capim-limão, orégano ou pimenta-do-reino no desenvolvimento in vitro de Colletotrichum gloeosporioides e de Fusarium moniliforme. Os extratos foram obtidos pela infusão de 60 g de cada planta medicinal em 200 mL de água fervente. Cada extrato aquoso foi fracionado em concentrações de 0, 5, 10 e 20% (p:v) e incorporado ao meio de cultivo BDA (batata-dextrose-ágar) antes da esterilização em autoclave. Posteriormente, um disco de 8 mm de diâmetro de micélio fúngico de cada patógeno foi transferido para o centro de placas de Petri. Após 24, 48 e 96 horas de incubação em câmara de crescimento a 22 ± 2 ºC e fotoperíodo de 12 horas avaliou-se o crescimento micelial de F. moniliforme e de C. gloesporioides. No último período de incubação, também se quantificou o número de conídios de cada fungo. Para o teste de germinação adicionou-se nas cavidades de placas de teste Elisa, uma alíquota de 40 µL de cada extrato nas concentrações de 0, 5, 10 e 20%, e outra alíquota, da suspensão de conídios de cada patógeno. Após 24 horas a 22 ± 2 ºC, no escuro, a germinação dos conídios foi paralisada com a adição de 20 µL de lactofenol; avaliou-se então a porcentagem de germinação de conídios. Os experimentos foram conduzidos no delineamento inteiramente casualizado em esquema fatorial 7 x 4 (extratos de plantas medicinais x concentrações) com quatro repetições. Para ambos os patógenos o extrato aquoso de alho e cravo-da-índia apresentaram maior ou total inibição do crescimento micelial, respectivamente, quando comparado com os demais extratos. Para C. gloeosporioides, o extrato de cravo-da-índia apresentou menor número de conídios em todas as concentrações testadas, e para o extrato de alho a 20%, também não foi observada a germinação de conídios. O extrato de alho foi eficiente em reduzir o número e a germinação dos conídios de F. moniliforme na concentração de 20%. Os extratos de alecrim, cravo-da-índia, orégano e pimenta-do-reino, nas maiores concentrações, tiveram efeito positivo na redução da produção de conídios deste mesmo fungo.

The objective of this study was to evaluate the influence of aqueous extracts of the medicinal plants rosemary, garlic, clove, sage, lemongrass, oregano and black pepper in the in vitro development of Colletotrichum gloeosporioides and Fusarium moniliforme. The extracts were obtained by infusing 60 g of each medicinal plant in 200 mL of boiling water. Each aqueous extract was fractionated in the concentrations of 0, 5, 10 and 20% (w:v) and incorporated into the PDA (potato dextrose agar) culture medium before sterilization by autoclaving. Later, an 8 mm diameter disc of each pathogen mycelium was transferred to the center of the Petri dishes. After 24, 48 and 96 hours of incubation in a growth chamber at 22 ± 2 ºC and a photoperiod of 12 hours, we evaluated the mycelial growth of F. moniliforme, and C. gloesporioides. In the last period of incubation, we quantified the production of conidia of each fungus. For the germination test, we added, into the wells of an ELISA test plates, a 40 µL aliquot of each extract at the concentrations of 0, 5, 10 and 20% and another aliquot of a suspension of conidia of each pathogen. After 24 hours at 22 ± 2 ºC in the dark, the germination of the fungi was stopped with the addition of 20 µL of lactophenol. Then, we evaluated the germination of conidia. The experiments followed a completely randomized 7 x 4 factorial design (medicinal plants x concentrations) with four replications. For both pathogens, the aqueous extract of garlic and clove showed a greater or total inhibition of the mycelial growth, when compared to the other extracts. For the C. gloeosporioides, the clove extract showed a lower number of conidia at all concentrations tested, and the garlic extract at 20% showed not conidial germination. The garlic extract was efficient to reduce the conidial number and germination of F. moniliforme at 20%. Extracts of rosemary, clove, oregano and black pepper, in the highest concentrations, had positive effect in reducing the production of spores of the same fungus.

Study in vitro of the impact of endophytic bacteria isolated from Centella asiatica on the disease incidence caused by the hemibiotrophic fungus Colletotrichum higginsianum.

Thirty-one endophytic bacteria isolated from healthy leaves of Centella asiatica were screened in vitro for their ability to reduce the growth rate and disease incidence of Colletotrichum higginsianum, a causal agent of anthracnose. Isolates of Cohnella sp., Paenibacillus sp. and Pantoea sp. significantly stimulated the growth rate of C. higginsianum MUCL 44942, while isolates of Achromobacter sp., Acinetobacter sp., Microbacterium sp., Klebsiella sp. and Pseudomonas putida had no influence on this plant pathogen. By contrast, Bacillus subtilis BCA31 and Pseudomonas fluorescens BCA08 caused a marked inhibition of C. higginsianum MUCL 44942 growth by 46 and 82 %, respectively. Cell-free culture filtrates of B. subtilis BCA31 and P. fluorescens BCA08 were found to contain antifungal compounds against C. higginsianum MUCL 44942. Inoculation assays on in vitro-cultured plants of C. asiatica showed that foliar application of B. subtilis BCA31, three days before inoculation with C. higginsianum MUCL 44942, significantly reduced incidence and severity of the disease. The role of endophytic bacteria in maintaining the apparent inactivity of C. higginsianum MUCL 44942 in C. asiatica grown in the wild is discussed.

Bioassay-directed isolation and identification of phytotoxic and fungitoxic acetylenes from Conyza canadensis.

Conyza canadensis (L.) Cronquist syn. (horseweed) is a problematic and invasive weed with reported allelopathic properties. To identify the phytotoxic constituents of the aerial parts, a systematic bioactivity-guided fractionation of the dichloromethane extract was performed. Three active enyne derivatives, (2Z,8Z)-matricaria acid methyl ester, (4Z,8Z)-matricaria lactone, and (4Z)-lachnophyllum lactone, were identified. The lactones inhibited growth of the monocot Agrostis stolonifera (bentgrass) and the dicot Lactuca sativa (lettuce) at 1 mg mL(-1), while the (2Z,8Z)-matricaria acid methyl ester was less active. In a dose-response screening of the lactones for growth inhibitory activity against Lemna paucicostata , (4Z)-lachnophyllum lactone was the most active with an IC50 of 104 µM, while the (4Z,8Z)-matricaria lactone was less active (IC50 of 220 µM). In a fungal direct bioautography assay, the two lactones at 10 and 100 µg/spot inhibited growth of the plant pathogenic fungi Colletotrichum acutatum , Colletotrichum fragariae , and Colletotrichum gloeosporioides . In a dose-response screening of the lactones against six different plant pathogenic fungi, (4Z,8Z)-matricaria lactone was more active than the commercial fungicide azoxystrobin on Col. acutatum , Col. fragariae , and Col. gloeosporioides at 30 µM and about as active as the commercial fungicide captan against Col. gloeosporioides , while (4Z)-lachnophyllum lactone was less active.

Synthesis and antifungal activities of carabrol ester derivatives.

Thirty-eight new ester derivatives of carabrol were designed, synthesized, and characterized by (1)H and (13)C NMR and HR-ESI-MS. Their antifungal activities against the fungal pathogen Colletotrichum lagenarium were evaluated using a spore germination assay. Of these 38 ester derivatives, 16 showed higher antifungal activity than that of carabrol and 7 showed higher antifungal activity than that of carabrone. It was found that the C-4 position of carabrol was a key position involving its antifungal activity, which showed the variation of 50% inhibition concentration (IC(50)) from 2.70 to 52.33 µg/mL. When substituted by the phenyl ring, the ester derivatives with electron-attracting groups showed higher activity than those with electron-donating ones. Two ester derivatives, carabryl 4-cynaobenzoate (II-17, IC(50) 2.70 µg/mL) and carabryl 4-isopropylbenzoate (II-27, IC(50) 2.82 µg/mL), showed only slightly lower antifungal activity than that of the positive control chlorothalonil (IC(50) 0.87 µg/mL) and have been identified as promising leads for development of new environmentally friendly fungicides.