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Acremonium implicatum is a seed-transmitted endophytic fungus that forms symbiotic associations with the economically significant tropical forage grasses, Brachiaria species. To take advantage of the endophyte's plant protective properties, we developed an efficient Agrobacterium-mediated transformation system for Acremonium implicatum, using green fluorescent protein (GFP) expression and vector pSK1019 (trpC promoter) or pCAMBIA1300 (CaMV35S promoter). We found that transformation efficiency doubled for both mycelial and conidial transformation as the co-cultivation period for Agrobacterium tumefaciens and Acremonium implicatum was increased from 48 to 72h. Significantly, optimal results were obtained for either mycelial or conidial transformation with Agrobacterium tumefaciens strain AGL-1 and vector pSK1019 under the control of the trpC promoter. However, mycelial transformation consistently generated a significantly higher number of transformants than did conidial transformation. The mitotic stability of the transferred DNA was confirmed by growing ten transformants in liquid and agar media for six generations. In all cases, resistance to the selection pressure (hygromycin B) was maintained. Fluorescence emission was retained by the transformants and also expressed in Brachiaria tissues from plants inoculated with GFP-transformed A. implicatum. This technology will help in the transfer and expression of agronomically important genes in host plants.

Brachiaria is a pan-tropical genus of grasses with about 100 species. The fungus Acremonium implicatum can develop an endophytic association that is mutually beneficial with Brachiaria species. We developed a polymerase chain reaction (PCR)-based method by first amplifying DNA from A. implicatum isolates using the Random amplified polymorphic DNA (RAPD) technique with arbitrary 10-mer primers. A 500-bp PCR product, amplified with primer OPAK-10 and common to A. implicatum isolates, was selected for further evaluation. The fragment was digoxygenin-labelled and used to probe a dot blot containing genomic DNA from isolates of A. implicatum and non-endophytic fungi, and from Brachiaria species free of endophytes. Strong signals were obtained only for DNA from A. implicatum isolates. This fragment was cloned and subsequently sequenced. Based on the sequence data, two primers were selected and synthesized: P1 (5'-TTCGAATGATAAGGCAGATC-3') and P4 (5'-ACGCATCCACTGTATGCTAC-3'). The primer pair amplified a single fragment of about 500 bp from DNA of A. implicatum isolates, whether from pure culture or in association with Brachiaria plants. No amplification product was detected in DNA from endophyte-free plants, pathogenic fungi, the bacterium Xanthomonas campestris pv. graminis, or non-pathogenic fungi associated with Brachiaria. This assay thus allows the precise and rapid detection of endophytes in Brachiaria plants and permits a differentiation between endophytic and non-endophytic fungi. [ABSTRACT FROM AUTHOR]

Brachiaria, predominantly an African genus, contains species, such as B. brizantha, an apomictic C[sub 4] grass, that are commercially important forage grasses in tropical America, where they now cover about 55 million hectares. From B. brizantha accession CIAT 6780, we isolated an endophytic fungus that may be economically significant. The fungus was identified as Acremonium implicatum (J. Gilman & E.V. Abott). 18S rDNA and ITS rDNA sequences were used to characterize isolates of the endophyte, and showed that they belonged to the Acremonium genus, being close to A. strictum and A. kiliense. Using the random amplified polymorphic DNA (RAPD) technique, involving arbitrary primers of 10 bases, we showed that the isolates were highly similar to each other. Antiserum produced from a monoconidial culture of A. implicatum isolated from B. brizantha 6780, differentiated the isolates consistently in line with the DNA data. When we compared endophyte-free with endophyte-infected B. brizantha CIAT 6780 plants, both artificially inoculated with the pathogenic Drechslera fungus, we found that the endophyte-infected plants had fewer and smaller lesions than did the endophyte-free plants. Sporulation of Drechslera sp. on artificially inoculated leaf sheath tissues was also much less on tissue infected with the endophyte.Key words: endophytes, Brachiaria, Acremonium, biological control.Brachiaria est un genre principalement Africain qui contient des espèces, telles que B. brizantha, une herbe C[sub 4] apomictique, représentant des herbes de fourrage commercialement importantes dans l'Amérique tropicale, où elles couvrent à ce jour environ 55 millions d'hectares. Nous avons isolé à partir de B. brizantha référence CIAT 6780 un champignon endophytique qui pourrait être commercialement important. Le champignon fut identifié comme étant Acremonium implicatum (J. Gilman & E.V. Abott). Les séquences des ADNr 18S et ADNr ITS furent utilisées afin de caractériser les isolats de l'endophyte. Les séquences démontrèrent qu'ils appartenaient au genre Acremonium, et étaient apparentés à A. strictum et à A. kiliense. En utilisant la technique du RAPD (ADN polymorphe amplifié au hasard), impliquant des amorces arbitraires de 10 bases, nous avons montré que les isolats étaient fort semblables entre eux. Un antisérum produit par une culture monoconidiale de A. implicatum isolé de B. brizantha 6780 a permis de différentier les isolats correctement, en harmonie avec les données d'ADN. Lorsque nous avons comparé les plants de B. brizantha 6780 infectés par l'endophyte avec celui dépourvu d'endophyte, les deux groupes ayant été inoculés avec le champignon pathogène Drechslera, nous avons découvert que les plants infectés avec l'endophyte avaient de plus petites et moins nombreuses lésions, comparativement aux plants sans endophyte. La sporulation de Drechslera sp. sur des tissus de gaine de feuille inoculées artificiellement était aussi diminuée de beaucoup chez les tissus infectés avec l'endophyte.Mots clés : endophytes, Brachiaria, Acremonium, contrôle biologique.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]

The pan-tropical grass genus Brachiaria comprises about 100 species, several of which are forages of economic importance, particularly in tropical America. Acremonium implicatum is a fungus that forms an endophytic association with at least some of these economically important grasses. To ascertain whether A. implicatum can be seed transmitted in Brachiaria species, we vegetatively propagated, under greenhouse conditions, 20 tillers from an endophyte-infected mother plant obtained from each of 14 Brachiaria hybrids and species. Ten tillers of each genotype were treated with the fungicide tebuconazole to eliminate the endophyte, and the other 10 were left untreated. Seeds were then harvested individually from all 20 of these genetically identical plants, germinated, and the seedlings grown. A previously developed polymerase chain reaction-based method used a pair of endophyte-specific primers to amplify a diagnostic 500-bp DNA fragment. The seedlings generated from seeds harvested from endophyte-infected plants also tested positive, whereas those from seeds of endophyte-free plants showed no amplification products. This is the first report of A. implicatum being transmitted through seeds of Brachiaria grasses. [ABSTRACT FROM AUTHOR]