Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.

Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.

Colonization of cashew plants by Lasiodiplodia theobromae: microscopical features.

Lasiodiplodia theobromae is a phytopathogenic fungus causing gummosis, a threatening disease for cashew plants in Brazil. In an attempt to investigate the ultrastructural features of the pathogen colonization and its response to immunofluorescence labeling, light, confocal and electron microscope studies were conducted on different severity scale patterns of diseased plants. Lasiodiplodia-antisera was checked for cross reactivity against common cashew plants fungi. Optical microscopy analysis revealed a longitudinally sectioned hyphae located within the xylem vessels, showing an extensive hyphal development in the secondary xylem tissue. SEM images demonstrated that the fungus was found in some asymptomatic samples, particularly within the xylem vessels as confirmed by the optical images. Symptomatic sample images showed an extensive distribution of the fungus along the secondary xylem, within the vessels, infecting xylem parenchyma. A closer look in the secondary xylem parenchyma reveals a heavy and profuse invasion of the cells with a distinguishable cell wall disintegration and fully hyphae dispersal. There was no reactivity of Lasiodiplodia-antisera against mycelial extracts of Colletotrichum gloeosporioides, Phomopsis anardii and Pestalotiopsis guepinii. Following incubation of sections with the polyclonal antisera, the hyphae were intensely and regularly labeled. Rays, vessels and parenchyma cells were the preferred pathway for L. theobromae colonization. Artificial infection provides the information that the vascular cylinder is undoubtedly employed and used by the fungus for hyphae distribution. Immunofluorescence assay employed in situ was applied and the polyclonal antisera produced was able to recognize the fungus and proved to be a sensitive technique to detect it.

First Report of Black Branch Dieback of Cashew Caused by Lasiodiplodia theobromae in Brazil.

Cashew nut (Anacardium occidentale) is one of the most important cash crops of northeastern Brazil. A new disease, named here as black branch dieback, caused by Lasiodiplodia theobromae, was observed causing serious damage on as many as 30% of the trees in some orchards in both coastal and inland semiarid cashew-growing areas of Ceará and Piauí states of Brazil, respectively. The disease symptoms are first observed as darkened, elongated lesions on stems near the branch apexes of herbaceous tissues. Gum exudation is common from lesions, which expand rapidly to affect the entire branch, leading to branch death. Diseased plants were collected, and L. theobromae was consistently isolated from canker tissues. Fresh mycelial disks of the fungus were used for artificial inoculation of healthy plants. Shoots of young cashew plants were inoculated on the apex by inserting a 3-mm plug taken from actively growing colonies on potato dextrose agar into an incision made with a sterile scalpel. Agar plugs with no mycelium were placed into incised plant shoots to serve as controls. Plants were incubated in a greenhouse at 28°C. Symptoms developed within 15 days after inoculation. Artificially inoculated plants showed symptoms similar to those that were naturally infected. L. theobromae was consistently reisolated from inoculated plants. The disease seems to occur throughout the year, but it spreads faster during the rainy season. A contagious disease pattern within the orchard was observed with a decreasing gradient from the orchard perimeter to the interior of the field, suggesting an external source of primary inoculum. All improved dwarf cashew clones were susceptible, but the newly released clone END-189 was the most susceptible. Black branch dieback may reduce tree growth, nut yield, and eventually cause plant death. Plant susceptibility is not related to its age however; only herbaceous tissues are vulnerable to natural infection. A similar disease on floral shoots of cashew caused by L. theobromae was reported by Olunloyo and Esuruoso in Nigeria (1). To our knowledge, this is the first report of L. theobromae causing branch dieback in cashew orchards in Brazil. Reference: (1) O. A. Olunloyo and O. F. Esuruoso. Plant Dis. 59:176, 1975.