J. E. F. Figueiredo

Genetic diversity among proso millet (Panicum miliaceum) biotypes assessed by AFLP technique

A tecnica de AFLP (Amplified Fragment Length Polymorphism) foi empregada para acessar a diversidade genetica entre tres biotipos domesticados e nove biotipos selvagens de proso millet dos Estados Unidos e do Canada. Oito combinacoes de primers detectaram 39 fragmentos polimorficos de DNA, e a estimativa da distância genetica entre os biotipos variou de 0,02 a 0,04. Colorado-Weld County de sementes pretas e Wyoming-Platte County foram os biotipos mais distintos de acordo com o indice de dissimilaridade. A analise de cluster por UPGMA revelou dois grupos distintos de proso millet mas sem nenhuma relacao geografica. Seis biotipos selvagens que exibiam algumas caracteristicas de plantas cultivadas foram agrupados juntamente com os biotipos domesticados de proso millet, enquanto os tres fenotipos tipicamente selvagens formaram outro grupo distinto por marcadores AFLP.

Molecular analysis of endophytic bacteria from the genus Bacillus isolated from tropical maize (Zea mays L.)

Endophytic bacteria play an important role in agriculture by improving plant performance and adaptation against biotic and abiotic stresses. In the present study molecular methods were used for identifying Bacillus endophytic bacteria isolated from Brazilian sweet corn. SDS-PAGE of wholecell protein extract of fortytwo isolates revealed a high number of scrutinable bands. Twenty-four isolates were identified in nine different groups of duplicated bacteria and eighteen were identified as unique. Some high-accumulated polipeptides with variable length were observed in almost isolates. Partial sequencing of 16S ribosomal gene revealed that all isolates are Bacillus sp. and among thirteen isolates with similar protein profiles, two were different strains. Among the forty-two isolates identified by rDNA sequencing, Bacillus subitilis and B. pumilus were the most frequenty species (15 and 12 isolates, respectively) followed by B. licheniformes (7 isolates), B. cereus (5 isolates) and B. amiloliquefascens (3 isolates). According to present results, SDS-PAGE technique could be used as a fast and cheap first tool for identifying interspecific variation in maize endophytic bacterial collections while rDNA sequencing could be applied for analyzing intraspecific variation among isolates with similar protein profile as well as for taxonomic studies.

Identificação através de PCR dos genes CryI de cepas de Bacillus thuringiensis Berliner eficientes contra a lagarta do cartucho, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

Fall armyworm, Spodoptera frugiperda (J. E. Smith), is the most important maize insect pest in Brazil and its damage can reduce yield up to 34%. The objective of this work was to utilize the PCR technique to Identify Bacillus thuringiensis (B.t) strains that control S. frugiperda in maize crops. Bioassays showed that 16 strains were very effective against S. frugiperda, 15 of them collected from soil samples and one (T09) obtained at the Institute Pasteur. The DNA of these strains were probed with cryI general primers and their proteins were analised by SDS-PAGE eletrophoresis. All strains present positive results for cryI genes. To further characterize these strains, specific cryI primers were employed. PCR technique showed that some strains harbour the same cryI genes. The only difference was the amplification of an unexpected fragment of approximately 160bp when a mixture of cryIB and cryID specific primers was used. Analysis by phase contrast microscope showed that crystal proteins produced by these strains were all bipyramidal crystals. Also, eltrophoretic analysis of proteins by SDS-PAGE showed the same protein banding pattern for most of the strains.

ETIOLOGY OF PHAEOSPHAERIA LEAF SPOT DISEASE OF MAIZE

Different fungal species and the bacterium Pantoea ananatis (Pa) have been reported as etiological agents of Phaeosphaeria leaf spot (PLS) disease. This work aimed at using molecular identification of the fungi and bacteria occurring in PLS and its etiology. Genomic DNAs from (i) pool of each of the four stages of PLS lesions; (ii) bacteria and fungi isolated from lesions of natural PLS (NPLS) and artificial injuries (AI); (iii) fungi isolated from lesions obtained from plants inoculated with Pa in greenhouse (GH) were used in PCR with universal primers for bacteria and fungal rRNA genes, and species-specific primers for Pa. Bacterial amplicons were observed at all stages of lesions and fungal amplicons in stages 3 and 4. Bacterial amplicons of pooled NPLS lesions were from Pa while fungal amplicons were from Phaeosphaeria sp. and Phoma sp. Bacteria from NPLS, GH and AI lesions were identified as Pa, Pa and Bacillus subtilis, respectively, while the fungi were Epicoccum nigrum, Leptosphaeria sacchari, Cochliobolus geniculatus, Pithomyces chartarum, Alternaria alternata, A. ricini, Gibberella intricans, G. fujikuroi, Phaeosphaeria sp., P. avenaria, Phoma sp., Phyllosticta sp., Sarocladium strictum, Glomerella graminicola, and Cochliobolus heterostrophus. Symptoms of PLS decreased by 90% with the use of oxytetracycline in foliar treatment of maize plants in the field, and its addition to culture medium completely inhibited the growth of Pa. The results strongly show that Pa is the causal agent of PLS disease and different species of opportunistic fungi appear late in the necrotic stages of lesions caused by Pa.

Novel insights into the early stages of infection by oval conidia of Colletotrichum sublineolum

Anthracnose, caused by Colletotrichum sublineolum Henn. ex Sacc. & Trotter, is one of the most important sorghum [Sorghum bicolor (L.) Moench] diseases in Brazil. This fungus exhibits conidial dimorphism, producing either falcate or oval conidia on solid and liquid media, respectively. We compared patterns of the initial infection events by these two types of conidia on sorghum leaves using light microscopy and scanning electron microscopy. The infection events during the first 24 h were similar for both oval and falcate conidia. Globose and melanized apressoria were formed at 24 h after inoculation (hai) regardless of the conidia type. Dense mycelium and oval conidia developed from germinated falcate conidia at 32 hai. Hyphal mass displaying acervuli filled with falcate conidia and surrounded by setae, developed from germinated oval conidia at 48 hai. Oval conidia were as capable as falcate conidia of infecting sorghum leaves. The inherent ability to grow faster and the easeness with which oval conidia can be produced in vitro as compared to falcate, make the former a preferred choice for studies on the C. sublineolum-sorghum interaction. It would be instructive to further investigate the potential role of the oval conidia in epidemics.

ETIOLOGY AND EPIDEMIOLOGICAL VARIABLES ASSOCIATED WITH MAIZE RESISTANCE TO WHITE SPOT DISEASE

SUMMARY Maize white spot disease (MWSD) has become most important in Brazil. With this study the susceptible maize hybrid DAS657 was artificially inoculated under greenhouse conditions with the bacterium Pantoea ananatis and the fungus Phaeosphaeria maydis, the two most likely candidate etiological agents of MWSD. Plants inoculated with P. ananatis reproduced the symptoms typical of MWSD, while several attempts to fulfill Koch’s postulates with P. maydis failed. The epidemiological variables associated with maize resistance to MWSD was assessed with eight simple hybrids, one double hybrid, one triple hybrid and five inbred lines evaluated in two trials in a randomized block design with three replicates in two planting times. The area under disease progress curve (AUDPC) and disease severity at the end of the epidemic (Y max ) were the epidemiological variables that better differentiated maize genotypes resistant to MWSD. One inbred line and three simple hybrids were highly resistant. Two inbred lines were identified as new sources of resistance and may be used in maize breeding programs.