and Teerasak Manupeerapan¹

1 Field Crop Research Institute, Department of Agriculture Bangkok, Thailand.

2. Department of Plant Pathology, Kasetsart University, Bangkok, Thailand.

…………………………………..

One hundred and seventeen isolates of Bipolaris maydis (Nisik. And Miyake) were collected from different maize planting areas throughout Thailand. The aim was to assess the genetic and pathogenic variability using culture characteristics, mating type, pathogenicity and AFLP analysis. The results show differences in the colony morphological characters, such as the colony, zonation of colour. From the mating type tested, it was found that 51 isolates are classified as mating type M1-1 and 66 isolates as M1-2, accounting for 43.6% and 56.4%, respectively.

Nineteen representative isolates were evaluated for pathogenic variability on 22 inbred lines of maize. Significant differences occurred between the isolates and the maize genotypes. The same virulent patterns were not found. Analysis of the AFLP data revealed a close relationship in B. maydis and this could be divided into two groups. Most of the isolates were clustered into one group, except for three that showed differences in their DNA patterns to others.

Southern Corn Leaf Blight that is caused by the fungus Bipolaris maydis (Nisik. and Miyake) is one of the most important maize diseases in Thailand. It is prevalent throughout the growing area in Thailand and causes yield and quality reductions. (Anchareesangas, 1998). Up until the present there has been no investigation into the biology and variability of this fungus. The planting environment, varieties used, together with type of fungicide used can lead to significant variation in the pathogen populations. In addition, the mode of reproduction of the fungus is also another important factor that shapes the pathogen’s genetic structure. The B. maydis is heterothallic meaning that mating type is controlled by a single gene with two alleles resulting in the recombination of hypha to produce the sexual reproductive organ.

Studies of population genetics and mode of reproduction of this pathogen are used to describe and quantify the genetic variations within and among the different populations. Such studies will yield information useful for more effective disease management. For example, if this pathogen shows high variation, then the use of a resistant variety derived either from a cultivar mixture or near-isogenic lines having many types of vertical resistance would be the most appropriate management strategy. Having knowledge of the mode of fungus reproduction within the population can be used to predict the possible changes in the genetics of the pathogen enabling more efficient control measures to be developed.

In recent years, plant pathologists have adopted the use of the molecular marker as a population genetic tool. Amplified fragment length polymorphisms (AFLP) have proved to be highly polymorphic and robust for use as markers. In contrast to virulence markers, molecular markers are assumed to be selectively neutral. The objective of this study was to evaluate the genetic variation of B. maydis in Thailand using morphological characters, mating type, and the ability to cause disease (pathogenicity).

Isolate and mating type test : Samples of B. maydis were collected from maize fields between year 1999-2000 and these were cultured and maintained on potato dextrose agar (PDA). A pair of unknown and known mating type were crossed on Sach’s medium. Pseudothecium was determined 3 week after crossing.

Pathogenicity test : Nineteen isolates were evaluated for pathogenicity on 22 maize inbred lines. Isolates of B. maydis were cultured as describe above. Conidial suspensions were prepared by washing the surfaces of 14-day-old cultures with sterile water and adjusting the final concentrations to approximately 2500 conidia per ml with the aid of hematocytometer. Inoculum was applied to the leaves of 3-week-old plants and after 5 days the length of the lesions were measured. All statistical analyses were done using ANOVA.

Cultural characteristics : The cultural feature of B. maydis isolates were evaluated on PDA medium. Mycelial plugs were placed in the centre of the petri-dishes and incubated at room temperature followed by exposure to a 12 hour-photoperiod, under near UV light. Colony colour and pattern of zonation were recorded after 7 days.

DNA extraction : Total DNA was extracted from freeze-dried mycelium according to the procedure of Vandemark et al. (2000). The DNA was resolved in deionised water and stored at 4 ^oC until required.

AFLP analysis : The protocol employed for AFLP analysis was a version of that proposed by Vos et al. (1995). In brief, a primary DNA template is prepared by digesting 500 ng of DNA with Eco RI and Tru 9I at 37 ^oC for 1 h. The digested DNA is then ligated to Eco RI and Mse I adapter for 3 h at 37 ^oC. The primers used for the pre-amplification step were Eco RI+A and Mse I+C, the pre-amplification reaction was diluted 1:10 in dH₂O prior to the second selective amplification. Selective amplification were done using the primer combinations of Eco RI+A and Mse I+CAT, Eco RI+A and Mse I+CAC, Eco RI+A and Mse I+CAG, Eco RI+AC and Mse I+C. The products were electrophoresed on 4.5% denaturing acrylamide gel, followed by a silver staining. Electrophoresis data were recorded as a binary, presence (1) or absence (0) matrix. Cluster analysis was performed using UPGMA in the SAHN procedure. (NTSYS-pc, version 1.7)

After 5 days of inoculation, two types of disease symptom were observed as follows: 1) necrotic lesion, and ; 2) chlorotic lesion. Other researcher has shown these to be certain levels of resistance in maize (Smith, 1975). Size and shape of symptoms are different among the different maize varieties, such as oval or rectangular shapes found along the leaf veins or eyespot-like-lesions. Some varieties also show a halo or purple ring that surround the lesions. The pathogenicity of the 19 isolates over 22 maize inbred lines were statistically different (P<0.01). Maize leaf lesions varied in length from averages of 3.12 to 5.17 mm. Some inbred lines, such as nei 402015, and nei 9204 (R ) are classified as resistant with average lesion length of 2.45 and 2.83 mm, respectively. Susceptible lines are, nei 402012, nei 4012013, nei 402014, nei 402019, and nei 402020 with average lesion lengths of between 5.00 to 5.84 mm. Based on the virulence pattern, no pair of isolates were found to have identical pattern (Table 1). The most virulent isolate, TF7 was found to cause disease on 15 lines of maize. From this study, we found that there was a wide variation between the fungus that we studied, therefore, the effective development of a maize variety that is resistance to southern corn leaf blight should be developed using the virulent isolates, together with a mix of isolates in order to test the disease interaction.

In the survey of B. maydis in Thailand, we found slight differences in colony colour uniformity characteristics. In some area, both mating types in the same place were found. Although, there is no evident on sexual reproduction found in nature. Both mating type in the same place may, however be caused by others genetic variation apart from sexual reproduction, such as para-sexual cycle. In this experiment, it was found that B. maydis show differences in disease severity on inbred lines tested. Cluster analysis generated using AFLP data found that most of pathogens showed closer genetic relationship than by using pathogenicity.