Relato de Rhizoctonia solani AG-4 HG I em crisântemo (Papiro Branco e Amarelo) e R. solani AG-4 HG III em gipsófila no estado de São Paulo, Brasil, e sua patogenicidade cruzada

Currently, anastomosis groups (AG) of Rhizoctonia sp. on chrysanthemum and occurrence of this fungus on gypsophila have not been reported in Brazil. However, in the present study, normal and cross pathogenicity and sequencing of ITS-5.8S rDNA regions were used to confirm the AG of isolate of Rhizoctonia sp. obtained from chrysanthemum (White Papyrus) and from gypsophila plants cultivated in Holambra / São Paulo, Brazil. After these tests, it was confirmed the report of Rhizoctonia solani AG-4 HG I on chrysanthemum (White and Yellow Papyrus) and R. solani AG-4 HG III on gypsophila in the São Paulo state, Brazil, and also their cross pathogenicity.

A origem de populações emergentes do patógeno da queima-da-folha (Rhizoctonia solani AG-1 IA) da Urochloa spp. na Amazônia e seu potencial de adaptação a outros agroecossistemas brasileiros

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Polissacarídeos da biomassa do basidiomiceto Rhizoctonia solani: extração, purificação e atividade biológica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The urochloa foliar blight and collar rot pathogen rhizoctonia solani AG-1 IA emerged in South America via a host shift from rice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comportamento do maracuajazeiro amarelo, variedade Afruvec, ante uma população de Fusarium solani, agente causal da podridão do colo

The objective of the present work was to verify the behavior of yellow passion fruit, Afruvec variety, in relation to a population of Fusarium solani, obtained from this crop. The experimental delineation was random blocks, containing 10 treatments [9 isolates and 1 control treatment], with 4 repetitions, each plot being represented by a vase containing a plant. A disk of culture medium colonized by each isolate of the fungus was inoculated in the wounded collar region of the plants of the Afruvec variety two months after sowing. The appraised parameters were: the pathogenicity, the incidence (number of dead plants) and the severity of the disease (length of the lesion in the collar region), until 60 days after inoculation. The Afruvec variety was susceptible to the fungus and also presented variability as to the severity of the disease and incidence in relation to the different isolates. The population of the fungus showed variability in regard to aggressiveness. In light of the evidence of genetic diversity in the F. solani population, it is also suggested, in the tests of selection of materials to the pathogen, that these materials should be evaluated in different places with a history of the disease or inoculation with a pool of isolates of the fungus, in order to know the wide resistance of the genotype to the pathogen.

Trichoderma harzianum expressed sequence tags for identification of genes with putative roles in mycoparasitism against Fusarium solani

The plant pathogen Fusarium solani causes a disease root rot of common bean (Phaseolus vulgaris) resulting in great losses of yield in irrigated areas of the Southeast and Midwest regions of Brazil. Species of the genus Trichoderma have been used in the biological control of this pathogen as an alternative to chemical control. To gain new insights into the biocontrol mechanism used by Trichoderma harzianum against the phytopathogenic fungus, Fusarium solani, we performed a transcriptome analysis using expressed sequence tags (ESTs) and quantitative real-time PCR (RT-qPCR) approaches. A cDNA library from T. harzianum mycelium (isolate ALL42) grown on cell walls of F. solani (CWFS) was constructed and analyzed. A total of 2927 high quality sequences were selected from 3845 and 37.7% were identified as unique genes. The Gene Ontology analysis revealed that the majority of the annotated genes are involved in metabolic processes (80.9%), followed by cellular process (73.7%). We tested twenty genes that encode proteins with potential role in biological control. RT-qPCR analysis showed that none of these genes were expressed when T. harzianum was challenged with itself. These genes showed different patterns of expression during in vitro interaction between T. harzianum and F. solani. (C) 2012 Elsevier Inc. All rights reserved.

Identification of differentially expressed genes from Trichoderma harzianum during growth on cell wall of Fusarium solani as a tool for biotechnological application

Background: The species of T. harzianum are well known for their biocontrol activity against many plant pathogens. However, there is a lack of studies concerning its use as a biological control agent against F. solani, a pathogen involved in several crop diseases. In this study, we have used subtractive library hybridization (SSH) and quantitative real-time PCR (RT-qPCR) techniques in order to explore changes in T. harzianum genes expression during growth on cell wall of F. solani (FSCW) or glucose. RT-qPCR was also used to examine the regulation of 18 genes, potentially involved in biocontrol, during confrontation between T. harzianum and F. solani. Results: Data obtained from two subtractive libraries were compared after annotation using the Blast2GO suite. A total of 417 and 78 readable EST sequence were annotated in the FSCW and glucose libraries, respectively. Functional annotation of these genes identified diverse biological processes and molecular functions required during T. harzianum growth on FSCW or glucose. We identified various genes of biotechnological value encoding to proteins which function such as transporters, hydrolytic activity, adherence, appressorium development and pathogenesis. Fifteen genes were up-regulated and sixteen were down-regulated at least at one-time point during growth of T. harzianum in FSCW. During the confrontation assay most of the genes were up-regulated, mainly after contact, when the interaction has been established. Conclusions: This study demonstrates that T. harzianum expressed different genes when grown on FSCW compared to glucose. It provides insights into the mechanisms of gene expression involved in mycoparasitism of T. harzianum against F. solani. The identification and evaluation of these genes may contribute to the development of an efficient biological control agent.

Mezzi innovativi nella difesa delle cucurbitacee da Fusarium solani f.sp. cucurbitae

Lo studio “Lotta biologica a Fusarium solani f.sp. cucurbitae su zucchino” si colloca nell’ambito della difesa integrata delle colture orticole dalle fitopatie fungine, in particolare quelle causate da patogeni ad habitat terricolo nei confronti dei quali è sempre più frequente il ricorso a mezzi di lotta diversi dai prodotti chimici. Interessante e innovativa appare la prospettiva di utilizzare microrganismi adatti a svilupparsi nel suolo, competenti per la rizosfera delle piante e con spiccate caratteristiche d’antagonismo verso i patogeni tellurici e di stimolazione delle difese sistemiche della pianta. Il marciume del colletto delle cucurbitacee, causato da diversi patogeni tra cui Fusarium solani f.sp. cucurbitae, rappresenta la principale malattia fungina di tipo tellurica che colpisce lo zucchino ed il melone nella Pianura Padana e che può portare a consistenti perdite produttive. Indagini condotte dal 2004 da parte del Diproval nell’areale bolognese, hanno evidenziato un’elevata frequenza del patogeno su zucchino coltivato soprattutto in tunnel. Considerata la carenza di conoscenze locali di F. solani f.sp. cucurbitae e di mezzi chimici di lotta efficaci, la ricerca svolta ha inteso approfondire la diagnosi della malattia e le caratteristiche biologiche degli isolati locali, e valutare la possibilità di utilizzare metodi biologici di lotta contro questo patogeno, nonché di studiare alcune delle possibili modalità d’azione di microrganismi antagonisti. Sono state pertanto prelevate, da zone diverse del Bolognese, campioni di piante di zucchino che presentavano sintomi di marciume del colletto ed è stato isolato il patogeno, che in base alle caratteristiche morfologiche macro e microscopiche, alle prove di patogenicità su diversi ospiti e a saggi biomolecolari, è stato identificato come Fusarium solani f. sp. cucurbitae W.C. Snyder & H.N. Hansen razza 1. Dagli isolati di campo sono state realizzate un centinaio di colture monosporiche venti delle quali sono state utilizzate per la prosecuzione delle prove. I venti ceppi sono stati saggiati per la loro patogenicità inoculandoli in terriccio sterile e con trapianto di giovani piante di zucchino. E’ risultata un’elevata variabilità del livello di virulenza tra i ceppi, stimata da 39% a 83% riguardo la gravità della malattia e da 61 a 96% per la frequenza di malattia. Sono state condotte prove di accrescimento miceliare che hanno evidenziato differenze tra i ceppi e tra gli esperimenti condotti a tre diverse temperature (17, 23 e 28°C) alla luce ed al buio. La crescita maggiore complessivamente è stata ottenuta a 23°C. I venti ceppi hanno anche mostrato di produrre, in vitro, vari livelli di enzimi di patogenesi quali cellulasi, poligalatturonasi, pectin liasi e proteasi. E’ stata evidenziata unan correlazione significativa tra attività cellulasica e pectin liasica con frequenza e gravità della malattia dei venti ceppi del patogeno. Le prove relative al contenimento della malattia sono state condotte in cella climatica. Sono stati considerati prodotti commerciali (Remedier, Rootshield, Cedomon, Mycostop, Proradix, Clonotry) a base rispettivamente dei seguenti microrganismi: Trichoderma harzianum ICC012 + T. viride ICC080, T. harzianum T22, Pseudomonas chlororaphis MA342, Streptomyces griseoviridis K61, P. fluorescens proradix DSM13134 e T. harzianum + Clonostachys rosea). I prodotti sono stati somministrati sul seme, al terreno e su seme+terreno (esperimenti 1 e 2) e in vivaio, al trapianto e vivaio+trapianto (esperimenti 3 e 4), riproducendo situazioni di pratico impiego. L’inoculazione del patogeno (un ceppo ad elevata patogenicità, Fs7 ed uno a bassa patogenicità, Fs37) è stata effettuata nel terreno distribuendo uno sfarinato secco di semi di miglio e cereali colonizzati dal patogeno. La malattia è stata valutata come intensità e gravità. I prodotti sono stati impiegati in situazioni di particolare stress, avendo favorito particolarmente la crescita del patogeno. Complessivamente i risultati hanno evidenziato effetti di contenimento maggiore della malattia nel caso del ceppo Fs37, meno virulento. La malattia è stata ridotta quasi sempre da tutti i formulati e quello che l’ha ridotta maggiormente è stato Cedomon. Il contenimento della malattia somministrando i prodotti solo nel terreno di semina o di trapianto è stato in generale quello più basso. Il contenimento più elevato è stato ottenuto con la combinazione di due tipologie di trattamento, seme+terreno e vivaio+trapianto. Le differenze tra i prodotti sono risultate più evidenti nel caso del ceppo Fs7. Per quanto riguarda lo studio di alcune delle modalità d’azione dei microrganismi contenuti nei formulati più efficaci, è stato verificato che tutti sono stati in grado di inibire, se pur in vario modo, la crescita del patogeno in vitro. Gli antagonisti più efficaci sono stati S. griseoviridis K61 (Mycostop) e P. fluorescens proradix DSM13134). I ceppi di Trichoderma, ed in particolare T.harzianum T22 (Rootshield), sono risultati i più attivi colonizzatori del substrato. Riguardo il fenomeno dell’antibiosi, il batterio P. fluorescens proradix DSM13134 ha mostrato di produrre i metaboliti non volatili più efficaci nel ridurre lo sviluppo del patogeno. Nelle condizioni sperimentali adottate anche i due ceppi di T. viride ICC080 e T. harzianum ICC012 hanno dimostrato di produrre metaboliti efficaci. Tali risultati, anche se relativi a prove in vitro, possono contribuire alla comprensione dei meccanismi dei microrganismi sul contenimento dell’infezione relativamente al rapporto diretto sul patogeno. E’ stato inoltre verificato che tutti i microrganismi saggiati sono dotati di competenza rizosferica e solo i batteri di endofitismo. Si conclude che, nonostante l’elevata pressione infettiva del patogeno che ha certamente influito negativamente sull’efficacia dei microrganismi studiati, i microrganismi antagonisti possono avere un ruolo nel ridurre l’infezione di F. solani f.sp. cucurbitae razza 1.

Estudio epidemiológico de la incidencia del hongo Alternaria alternata en la isla de Gran Canaria y rentabilidad clínica del diagnóstico de su alérgeno mayor Alt a 1

Programa de doctorado: Avances en medicina interna

Study of Italian isolates of Alternaria spp.: molecular and morphological characterization and pathogenesis on apple tree

It was decided to carry out a morphological and molecular characterization of the Italian Alternaria isolatescollected from apple , and evaluate their pathogenicity and subsequently combining the data collected. The strain collection (174 isolates) was constructed by collecting material (received from extension service personnel) between June and August of 2007, 2008, and 2009. A Preliminary bioassays were performed on detached plant materials (fruit and leaf wounded and unwounded), belonging to the Golden cultivar, with two different kind of inoculation (conidial suspension and conidial filtrate). Symptoms were monitored daily and a value of pathogenicity score (P.S.) was assigned on the basis of the diameter of the necrotic area that developed. On the basis of the bioassays, the number of isolates to undergo further molecular analysis was restricted to a representative set of single spore strains (44 strains). Morphological characteristics of the colony and sporulation pattern were determined according to previous systematic work on small-spored Alternaria spp. (Pryor and Michaelides, 2002 and Hong et al., 2006). Reference strains (Alternaria alternata, Alternaria tenuissima, Alternaria arborescens and four Japanese strains of Alternaria alternata mali pathotype), used in the study were kindly provided by Prof. Barry Pryor, who allows a open access to his own fungal collection. Molecular characterization was performed combining and comparing different data sets obtained from distinct molecular approach: 1) investigation of specific loci and 2) fingerprinting based on diverse randomly selected polymorphic sites of the genome. As concern the single locus analysis, it was chosen to sequence the EndoPG partial gene and three anonymous region (OPA1-3, OPA2- and OPa10-2). These markers has revealed a powerful tool in the latter systematic works on small-spored Alternaria spp. In fact, as reported in literature small-spored Alternaria taxonomy is complicated due to the inability to resolve evolutionary relationships among the taxa because of the lack of variability in the markers commonly used in fungi systematic. The three data set together provided the necessary variation to establish the phylogenetic relationships among the Italian isolates of Alternaria spp. On Italian strains these markers showed a variable number of informative sites (ranging from 7 for EndoPg to 85 for OPA1-3) and the parsimony analysis produced different tree topologies all concordant to define A. arborescens as a mophyletic clade. Fingerprinting analysis (nine ISSR primers and eight AFLP primers combination) led to the same result: a monophyleic A. arborescens clade and one clade containing both A. tenuissima and the A. alternata strains. This first attempt to characterize Italian Alternaria species recovered from apple produced concordant results with what was already described in a similar phylogenetic study on pistachio (Pryor and Michaelides, 2002), on walnut and hazelnut (Hong et al., 2006), apple (Kang et al., 2002) and citurus (Peever et al., 2004). Together with these studies, this research demonstrates that the three morphological groups are widely distributed and occupy similar ecological niches. Furthermore, this research suggest that these Alternaria species exhibit a similar infection pattern despite the taxonomic and pathogenic differences. The molecular characterization of the pathogens is a fundamental step to understanding the disease that is spreading in the apple orchards of the north Italy. At the beginning the causal agent was considered as Alteraria alternata (Marshall and Bertagnoll, 2006). Their preliminary studies purposed a pathogenic system related to the synthesis of toxins. Experimental data of our bioassays suggest an analogous hypothesis, considering that symptoms could be induced after inoculating plant material with solely the filtrate from pathogenic strains. Moreover, positive PCR reactions using AM-toxin gene specific primers, designed for identification of apple infecting Alternaria pathovar, led to a hypothesis that a host specific toxin (toxins) were involved. It remains an intriguing challenge to discover or not if the agent of the “Italian disease” is the same of the one previously typified as Alternaria mali, casual agent of the apple blotch disease.

Biogeografía de especies de Fusarium en el litoral mediterráneo de España

El trabajo presentado estudia la presencia de Fusarium oxysporum, F.solani(sensulato), F. equiseti y F.acuminatum en puntos del litoral de Almería, Alicante, Gerona e Islas Baleares (Menorca, Ibiza, Espalmador). Se analizaron tanto arenas de las playas (zonas intermareal y supramareal) como fondos marino situados a 27,9 y 7,2 metros de profundidad en Almería y a 10 m de profundidad en las Islas Baleares. Exceptuando el litoral de Gerona, en el resto de los enclaves se presentaron varias especies de Fusarium que se aislaron de las arenas de las playas, confirmando así resultados obtenidos con anterioridad. Lo más novedoso fue encontrar especies de Fusarium a diferentes profundidades marinas. En Almería F.oxysporum y F.equisti se aislaron a 27,9 y7,2 m profundidad. F. acuminatum se aisló de la muetra recogida a 27m de profundidad. En las islas Baleares, a10m de profundidad, se aislaron F. oxysporum, F. solani (sensulato), F.equiseti y F.acuminatum. El efecto antrópico, el comportamiento como "airborne" o los arrastres de aguas por las ramblas y torrentes podría explicar la presencia de estas especies en los hábitats mencionados. La permanencia de estas especies en los hábitats mencionados, especialmente en la zona intermareal de las playas y en los fondos marinos donde soportan elevadas presiones osmóticas por la alta salinidad del agua del mar Mediterráneo, permitirá estudios específicos sobre el comportamiento de estos hongos en medios muy salinos. Otros hongos aislados de arenas de playa y fondos marinos fueron: Acremonium, Alternaria, Aspergillus, Cladosporium, Dreschlera, Gliocladium Humicola, Penicillium, Phialophora, Rhizopus, Stemphylium, Trichoderma, Trichocladium y Ulocladium. Muchos de ellos fueron aislados del fondo marino, testimoniando así que estos hábitats no son exclusivos de Fusarium.

The interactive effects of temperature and osmotic potential on the growth of marines isolates of Fusarium solani

The mycelial growth of 18 Fusarium solani strains isolated from sea beds of the south-eastern coast of Spain was tested on potato-dextrose agar adjusted to different osmotic potentials with either KCl or NACl (-1.50 to -144.54 bars) in 10ºC intervals ranging from 15 to 35ºC. Fungal growth was determined by measuring colony diameter after 4 days incubation. Mycelial growth was maximal at 25ºC. The quantity and frequency pattern of mycelial growth of F. solani differ significantly at 15 and 25ºC, with maximal occurring at the highest water potential tested (-1.50 bars); and at 35ºC, with a maximal mycelial growth at -13.79 bars. The effect of water potential was independent of salt composition. The general growth pattern of F. solani showed declining growth at potentials below -41.79 bars. Fungal growth at 35ºC was always higher than that growth at 15ºC, of all the water potentials tested. Significant differences observed in the response of mycelia to water potential and temperature as main and interactive effects. The viability of cultures was increasingly inhibited as the water potential dropped, but some growth was still observed at -99.56 bars. These findings could indicate that marine strains of F. solani have a physiological mechanism that permits survival in environments with low water potential. The observed differences in viability and the magnitude growth could indicate that the biological factors governing potential and actual growth are affected by osmotic potential in different ways.