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Endophytic fungi live inside plants as symbionts for at least one period of their life cycle. They play an important role in plant defence and could be promising sources of valuable natural bioactive products. The present study aimed to select endophytic fungi antagonistic to Microcyclus ulei, the agent of South American Leaf Blight (SALB) responsible for the weak development of rubber plantations in Latin America. Endophytic fungi were isolated from leaves of three cultivars (FX3864, CDC312, MDF180) of Hevea brasiliensis (the rubber tree) with distinct resistance levels to the attack by Microcyclus ulei. Leaf samples were collected from a 5,000 ha area of the Michelin plantations (Igrapiúna, Bahia, Brazil). A total of 435 colonies of endophytic fungi were isolated, purified, and preserved in sterilized distilled water. Lyophilized culture filtrates obtained from 88 fungal isolates, grown in liquid malt extract medium, were tested in vitro at concentrations of 50% and 12.5% for their inhibitory potential against the germination of M. ulei PMB26 conidia. Thirteen filtered cultures of endophytic fungi that presented inhibitory activity above 80% (at the concentration 12.5%) were characterized taxonomically by sequencing the ITS regions of rDNA, revealing the following taxa: Fusarium sp., Gibberella sp., Glomerella cingulata, Microsphaeropsis sp., Myrothecium sp., Pestalotiopsis sp. and Phomopsis sp. Three taxa were detected only in the resistant cultivars: Microsphaeropsis sp., and Pestalotiopsis sp. in MDF180, and Myrothecium sp. in CDC312. The present work constitutes the first study showing inhibitory activity of rubber tree endophytic fungi on Microcyclus ulei. These results provide an encouraging basis for the identification of new bioactive molecules for an effective control of South American leaf blight.

The components of genetic resistance from the Hevea brasiliensis cultivar MDF 180 against South American Leaf Blight (SALB) caused by Microcyclus ulei were investigated by QTL mapping. MDF 180 has already been described as a cultivar with a high level of partial and long-lasting resistance. The resistance of progeny individuals from a cross between a susceptible cultivar and MDF 180 was assessed both under controlled conditions of inoculation by three M. ulei isolates and under natural infection in a field trial. Genetic maps of the two parents of this progeny were mainly established based on microsatellites and AFLP markers. No resistance QTL were found in the susceptible parent. In the resistant parent, we identified a qualitative gene responsible for the resistance against isolates from French Guiana and a major quantitative resistance factor determining the resistance against isolates from the state of Bahia (Brazil). The qualitative resistance gene was denominated M15md and was located in the linkage group g15. Four minor resistance QTLs were also identified, two of which showed an epistatic interaction with M15md. The durability of the resistance of MDF 180 is discussed in light of these data.

RESUMOO fungo Microcyclus ulei, causador do mal-das-folhas da seringueira (Hevea brasiliensis), é o maior responsável pelo insucesso da heveicultura nas áreas tradicionais de cultivo no Brasil. Com o objetivo de conhecer melhor a diversidade desse patógeno foram estudados 50 isolados, obtidos nos anos 1997 e 1998, de uma área de 5.000 ha de seringueira da "Plantações Michelin da Bahia", no Sudeste da Bahia, através do tipo de reação de 12 clones de seringueira. As inoculações foram realizadas na face inferior dos folíolos jovens, repetidas três ou mais vezes em plantas diferentes, em câmara úmida com umidade superior a 95% e temperatura variando de 23 a 26 ºC. Após 12 dias efetuaram-se as avaliações, utilizando-se uma escala de notas de 1 a 6, que mede a virulência e a intensidade de esporulação. Palavras-chave adicionais: virulência, mal das folhas da seringueira. ABSTRACT Variability of Microcyclus ulei isolates from Southeast BahiaSouth American Leaf Blight (SALB) of Hevea rubber (Hevea brasiliensis) tree, a disease caused by the fungus Microcyclus ulei, is the principal reason for the rather poor development of natural rubber production in Brazil. To obtain a more accurate knowledge of the diversity of this pathogen, 50 fungus isolates originated from the "Plantações Michelin da Bahia" were studied in 1997 and 1998, by observing the host's reaction type when inoculated on 12 different rubber clones. Inoculations were performed on the lower surface of young leaflets, with at least three replications in different plants and days, in an inoculation chamber with relative humidity superior to 95% and temperature between 23 and 26 °C. Evaluation of symptoms was carried out 12 days after inoculation, using a 1 to 6 scale which measured virulence and sporulation intensity. Thirty-six perfils of virulence were identified among the 50 tested isolates, and 21 showed virulence on more than nine clones while none showed virulence on all 12 different clones. The aggressiveness of isolates, evaluated through the intensity of sporulation, varied significantly from one clone to another. Each isolate usually presented a high degree of aggressiveness to some clones and a low degree to to others, with the exception of isolates FTP 11 and FTP 44 which always displayed a low degree of aggressiveness. For example, some isolates showed higher aggressiveness to Hevea benthamiana clones, while others was more aggressive to H. brasiliensis clones. Four isolates seem to be specific to clone FX 2784. 502Fitopatol. bras. 28(5), set -out 2003 INTRODUÇÃOA produção de borracha natural no Brasil em 1996, segundo dados do IBAMA, apresentados por Hoelz (1999), foi de 53.438 t, ou seja, 43% da demanda nacional, num país que apresenta condições edafo-climáticas altamente favoráveis ao desenvolvimento da heveicultura e dispõe de milhões de hectares para plantio.O insucesso da heveicultura (Hevea spp.) nas áreas tradicionais de cultivo no Brasil deve-se, principalmente, a ocorrência do mal das folhas da seringueira, causado pelo Microcyclus ulei, ...

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BackgroundSouth American leaf blight (SALB) of rubber has been the main constraint to production in its neotropical centre of origin since commercial plantations were first established. The fungal causal agent was identified and described more than a century ago but its precise placement within the Ascomycota still remains uncertain. Indeed, such is the ambiguity surrounding the pathogen that each of the spore morphs would, according to their present classification, be placed in different ascomycete families: the Microcyclus sexual morph in the Planistromellaceae and the two purported asexual morphs - Fusicladium and Aposphaeria – in the Venturiaceae and Lophiostomataceae, respectively. Given the historical importance of the fungus and the ever-menacing threat that it poses to rubber production in the Palaeotropics – and, thus to the rubber industry and to the global economy – its phylogeny, as well as its biology, should be resolved as a matter of urgency.Methods and ResultsHere, six genomic regions (LSU rRNA, mtSSU, MCM7, EF-1α, Act and ITS) were used for reconstructing the molecular phylogeny of the SALB fungus based on material collected throughout Brazil. The analyses support the classification of the fungus in the family Mycosphaerellaceae s. str. (Capnodiales, Dothideomycetes) and place it firmly within the clade Pseudocercospora s. str., now accepted as one of the distinct genera within Mycosphaerellaceae. The new combination Pseudocercospora ulei is proposed and the life cycle of the fungus is confirmed, based on both experimental and phylogenetic evidence, with the Aposphaeria morph shown to have a spermatial rather than an infective-dispersal function.ConclusionsBecause the phylogeny of the SALB fungus has now been clarified, new insights of its epidemiology and genomics can be gained following comparison with closely-related, better-researched crop pathogens.

Research Summary• Genetic resistance components of the Hevea brasiliensis × H. benthamiana RO 38 cultivar to Microcyclus ulei disease were investigated by inoculating isolates which succeeded in partially or completely infecting genotypes of a mapping population.• Progeny of a cross between RO 38 and a susceptible cultivar was inoculated under controlled conditions with three isolates and scored for two resistance traits. Interval mapping and a nonparametric test were used to detect resistance quantitative trait loci (QTLs).• Eight significant QTLs were detected, all of them inherited from the interspecific parent. Among these QTLs, only one contributed to the partial resistance against a highly pathogenic isolate, and no QTL was detected for resistance against the most pathogenic isolate. As an unexpected result, a single isolate can thus completely bypass this polygenic resistance.• This complex situation, where no clear relationship can be established between number of resistance factors and qualitative vs quantitative or partial vs complete resistance, is discussed and suggestions as to the detection of new and sustainable resistance sources are proposed.