Development of PCR-RFLP Technique for Identify Several Members of Fusarium incarnatum-equiseti Species Complex and Fusarium fujikuroi Species Complex.

Fusarium incarnatum-equiseti species complex (FIESC) contain over 40 members. The primer pair Smibo1FM/Semi1RM on the RPB2 partial gene has been reported to be able to identify Fusarium semitectum. The F. fujikuroi species complex (FFSC) contains more than 50 members. The F. verticillioides as a member of this complex can be identified by using VER1/VER2 primer pair on the CaM partial gene. In this research, the Smibo1FM/Semi1RM can amplify F. sulawesiense, F. hainanense, F. bubalinum, and F. tanahbumbuense, members of FIESC at 424 bp. The VER1/VER2 can amplify F. verticillioides, F. andiyazi, and F. pseudocircinatum, members of FFSC at 578 bp. Polymerase chain reaction-restriction fragment length polymorphism by using the combination of three restriction enzymes EcoRV, MspI, and HpyAV can differentiate each species of FIESC used. The two restriction enzymes HpaII and NspI can distinguish each species of FFSC used. The proper identification process is required for pathogen control in the field in order to reduce crop yield loss.

Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia.

<b>Background and Objective:</b> Coffee leaf rust disease caused by <i>Hemileia vastatrix</i> resulted in high yield loss and difficult to control. Several chemical fungicides have been used to control this disease. However, the effectiveness of chemical control is low, so it is necessary to find other methods such as biological control. <i>Lecanicillium</i> spp. is well-known as mycoparasite on <i>H. vastatrix</i> uredospores but the study in Indonesia is still limited. This study aimed to collect and investigated the genetic variability of <i>Lecanicillium</i> spp. at various coffee plantations in Indonesia. <b>Materials and Methods:</b> Samples of <i>Lecanicillium </i>spp. were collected from 20 districts in 7 provinces throughout Indonesia. Morphology of colony and conidia were identified by visual examination and by viewed under the light microscope. Genetic variability was conducted using Rep-PCR and clustered with UPGMA. <b>Results:</b> Morphological observation in this study revealed all isolates collected from uredospores of <i>H. vastatrix</i> were similar with <i>Lecanicillium </i>spp. Genetic variability analysis clustered the 80 isolates into eight clusters with their specific characters. <b>Conclusion:</b> Morphological identification in this study showed that 80 isolates of mycoparasite on <i>H. vastatrix</i> belong to <i>Lecanicillium</i> spp. Further study using the molecular technique is needed to identity the species of <i>Lecanicillium</i>.

Resistance Response of Chilli (Capsicum annuum L.) F1 to Fusarium oxysporum Involves Expression of the CaChi2 Gene.

Cross-breeding is a method of producing progeny with better resistance to pathogens. Resistance to pathogens usually involves pathogenesis-related (PR) proteins. Class II chitinase is an example of a defensive PR protein in plants. The class II chitinase in chilli is coded by the CaChi2 gene. In this study, we crossed susceptible with resistant chilli cultivars, analysed the F1 resistance response against pathogenic F. oxysporum, and analysed the level of CaChi2 gene expression in the F1. Data were collected using disease severity index (DSI) determination and gene expression analysis by qRT-PCR (quantitative Reverse Transcriptase Polymerase Chain Reaction). Results showed that the DSI of F1 was not significantly different from the resistant ancestor. The relative CaChi2 expression level of F1 was higher than the susceptible ancestor but not significantly different from the resistant ancestor. We concluded that the F1 can be categorised as resistant to F. oxysporum, and the CaChi2 gene is involved in the molecular defense response.

Heterokaryon formation in Thanatephorus cucumeris (Rhizoctonia solani) AG-1 IC.

Approximately 50 single-basidiospore isolates (SBIs) obtained from each of 16 field isolates of Thanatephorus cucumeris AG-1 IC were examined for heterokaryon formation. All SBIs obtained from each field isolate were divided into two mating groups (SBIs-M1 and SBIs-M2), and tufts of mycelia were formed in the contact zone between colonies of paired SBIs-M1 and -M2 based on 0.5 % charcoal agar medium. Tufts were produced from all possible pairing between SBIs from non-parental field isolates. Hyphal anastomosis reactions indicated no cell death and random cell death at the contact cell, and was not related to tuft formation. AFLP phenotypes of SBIs from each field isolate were not identical to each other and were different from their parental field isolate. AFLP phenotypes of the tuft isolates formed from SBIs-M1 and SBIs-M2 from each field isolate were heterokaryotic. Moreover, several SBIs also formed tufts with their parental and non-parental field isolates. AFLP phenotypes of these tuft isolates suggested that they were all heterokaryotic. Results of these experiments suggest that T. cucumeris AG-1 IC is heterothallic and bipolar, and that genetic exchange can occur between homokaryotic and heterokaryotic isolates (Buller phenomenon).

New Anastomosis Groups, AG-T and AG-U, of Binucleate Rhizoctonia spp. Causing Root and Stem Rot of Cut-Flower and Miniature Roses.

ABSTRACT Root and stem rot of cut-flower roses (Rosa spp.) was observed in commercial glasshouse-grown roses in 10 prefectures of Japan from 1998 through 2001. Binucleate-like Rhizoctonia spp. were isolated mainly from the disease plants. In all, 670 isolates were divided into two types based on cultural appearance; 168 isolates of light brown to brown type and 502 isolates of whitish type. A hyphal anastomosis reaction using representative isolates from each type revealed that the light brown to brown type belonged to anastomosis group G (AG-G), whereas the whitish type (AG-CUT) failed to anastomose with tester strains of binucleate Rhizoctonia AG-A through AG-S. Neither isolates of AG-G nor AG-CUT anastomosed with tester strains of a previously reported unknown AG (AG-MIN) of binucleate Rhizoctonia spp. collected from miniature roses. In pathogenicity tests, randomly selected isolates of the three groups caused root and stem rot on cut-flower and miniature roses. To differentiate AG-CUT and AG-MIN from known AGs of binucleate Rhizoctonia spp., restriction fragment length polymorphism (RFLP) and sequence analyses of a ribosomal (r)DNA internal transcribed spacer (ITS) region were conducted. Among the eight restriction enzymes used, HaeIII produced DNA banding patterns for AG-CUT that differed from those of tester strains and AG-MIN. Additionally, restriction profiles of AG-MIN differed from those of all tester strains. AG-G isolates from cut-flower roses had the same RFLP pattern as the tester strains of AG-G. Based on the results of hyphal anastomosis and RFLP and sequence analysis of an rDNA-ITS region, we propose that AG-CUT be designated AG-T and AG-MIN be designated AG-U, two new AGs of binucleate Rhizoctonia spp. The phylogenetic tree based on the sequence data of the rDNA-ITS region showed that isolates of AG-MIN were in a distinct clade from other AGs, whereas isolates of AG-CUT were in the same clade as those of AG-A. More detailed phylogenetic analysis besides rDNA-ITS region might be necessary for AG classification of binucleate Rhizoctonia spp.