Achmadi Priyatmojo

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.

Characterization of a New Subgroup of Rhizoctonia solani Anastomosis Group 1 (AG-1-ID), Causal Agent of a Necrotic Leaf Spot on Coffee.

ABSTRACT A new foliar disease on coffee leaves was observed in Mindanao, Philippines, in 1996. The symptoms appeared as large circular or irregularly shaped necrotic areas with small circular necrotic spots (1 mm or less in diameter) usually found around the periphery of the large necrotic areas. Rhizoctonia solani was consistently isolated from these diseased coffee leaves. Isolates obtained were multinucleate (3 to 12 nuclei per hyphal cell), had an optimum temperature for hyphal growth at 25 degrees C, prototrophic for thiamine, and anastomosed with tester isolates belonging to R. solani anastomosis group 1 (AG-1). Mature cultures on potato dextrose agar (PDA) were light to dark brown. Sclerotia, light brown to brown, were formed on the surface of PDA and covered the whole mature colony culture. Individual sclerotia often aggregated into large clumps (3 to 8 mm in diameter) and their color was brown to dark brown. In pathogenicity tests, isolates from coffee caused necrotic symptoms on coffee leaves, whereas isolates of AG-1-IA (not isolated from coffee), 1-IB, and 1-IC did not. The results of analyses of restriction fragment length polymorphism of ribosomal DNA internal transcribed spacer, random amplified polymorphism DNA, and fatty acid profiles showed that R. solani isolates from coffee are a population of AG-1 different from AG-1-IA, 1-IB, and 1-IC. These results suggest that R. solani isolates from coffee represent a new subgroup distinct from AG-1-IA, 1-IB, and 1-IC. A new subgroup ID (AG-1-ID) is proposed.

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).

Characterization of Rhizoctonia spp. causing root and stem rot of miniature rose

Root and stem rot of miniature rose (Rosa hybrida L.) was observed in commercial glasshouse-grown roses in Gifu prefecture, Japan, during the summer and fall of 1997 and 1998. One hundred and fifty-three isolates of Rhizoctonia spp. were obtained from infected roots and stems. Of the 153 isolates, 9 had binucleate and 144 had multinucleate vegetative hyphal cells. Binucleate Rhizoctonia failed to anastomose with tester isolates of anastomosis groups (AG)-A through -S (not including AG-J and AG-M). Of 144 isolates identified as R. solani, 83.3% were AG 2-2 IIIB and 16.7% were AG 4 HG-I. Five isolates from each group caused severe rot and mortality on cuttings during rooting. Pathogenicity of Rhizoctonia spp. varied on three different ages of miniature roses cv. Silk. Isolates of AG 4 HG-I caused root and stem rot and mortality on 15-, 25-, and 40-day-old plants, whereas isolates of AG-2-2 IIIB caused root and stem rot and mortality on 15- and 25-day-old plants, but light root rot on 40-day-old plants. Isolates of binucleate Rhizoctonia caused root and stem rot and mortality only on 15-day-old plants.

Activities of plant cell wall-degrading enzymes by bacterial soft rot of orchid

Soft rot by bacterial pathogens is one of the most widespread and destructive diseases on various plants including orchids throughout the world. The pathogenicity of the pathogens is reported to be mainly determined by massive production of plant cell wall-degrading enzymes (PCDE). In the previous work, we have isolated 20 isolates of bacterial soft rot from orchids collected in Yogyakarta Special Region and West Java province, Indonesia. In this study, we further confirmed them as pathogens by hypersensitive reaction assay on tobacco leaves followed by pathogenicity test on Phalaenopsis sp. The production of four major PCDE by qualitative plate assays including pectate lyase, polygalacturonase, cellulase and protease was also evaluated. Even though all the isolates were able to initiate soft rot symptom, our results showed two distinct groups which clustered as producing and non-producing PCDE. The 16S rDNA analysis revealed that the isolates belonged to the genera Pectobacterium, Klebsiella, Serratia, Enterobacter, Citrobacter, Providencia and Pseudomonas.

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.

Systemic inducing resistance against late blight by applying antagonist Trichoderma Viride

Biological control by Phytophthora infestans (Mont.) de Bary, a pathogen causing late blight disease on potatoes, has attracted scholars as an environmentally alternative preference to chemical pesticide. Some species of Trichoderma spp. has proven to be capable of triggering resistance mechanism of plant against fungal pathogens. The aim of this research was to analyze the effect of Trichoderma viride SP1 suspension to induced systemic resistance on potato plant as showing glucanase activity and total phenol content. The method used was Completely Randomized Design with six treatments and seven times repeated. Variables observed were total phenol content and total crop. The result showed that glucanase activity and total phenol content in plants treated with Trichoderma viride and fungicide (1587 μg/g and 1934 μg/g) significantly increased. In addition, T. viride enhanced induced systemic resistance on potato plant and increased potato yield.

Induction of Resistance Using Trichoderma spp. and Penicillium sp. against Banded Leaf and Sheath Blight (BLSB) Caused by Rhizoctonia solani in Maize

Banded leaf and sheath blight (BLSB), caused by Rhizoctonia solani, is an important disease in maize. In this study, Trichoderma spp. and Penicillium sp. were used to control the disease. Experiments were conducted with five treatments: control (no treatment) (R0); R. solani inoculation (R1); Trichoderma spp. and R. solani inoculation (R2); Penicillium sp. and R. solani inoculation (R3); and combined Trichoderma spp., Penicillium sp., and R. solani inoculation (R4). The results showed that the heights of maize plants treated with R3, R2, or R4 did not differ significantly in comparison with R0 treatment but did differ significantly in comparison with R1 treatment. The numbers of leaves in maize plants treated with R4, R2, or R3 differed significantly in comparison with R0 and R1 treatment. The stem girths of maize plants treated with R2, R3, or R4 did not differ significantly in comparison with R0 treatment, but a significant difference was observed in comparison with R1 treatment. Peroxidase enzyme activity with R0, R2, R3, or R4 treatment was increased at 4 days and 8 days after inoculation; on the other hand, enzyme activity with R1 treatment was increased only at 4 days after inoculation and was then decreased at 8 days after inoculation. The intensity of disease ratings with treatments R0, R1, R2, R3, and R4 were about 2%, 28%, 10%, 9%, and 5%, respectively.

Identifikasi Morfologi Beberapa Spesies Jamur Fusarium

The research was conducted to study the morphological difference based on macroscopic and microscopic appearance of several Fusarium spp. Fusarium spp. isolates were propagated onto Potato Dextrose Agar (PDA) by using single-spore method. All isolates were observed macroscopically and microscopically to determine colony appearance, colony growth diameter and formation of macroconidia, microconidia and conidiophores. The results showed that colony appearance of all isolates was similar. Therefore they could not clearly be differentiated by one to another. On the other hand, microscopic observation showed that there was different conidiophore morphology of F. oxysporum and other Fusarium spp. Microscopic morphology among F. oxysporum isolates were difficult to differentiate. Microconidia were produced in false-head which was the characteristic feature of most F. oxysporum. In conclusion, microscopic morphology observation could only be able to differentiate Fusarium spp. isolates at species level, but not to formae speciales. Penelitian ini bertujuan untuk mengetahui perbedaan morfologi berdasarkan morfologi makroskopis dan mikroskopis beberapa isolat Fusarium spp. Isolat Fusarium spp. diperbanyak pada medium Potato Dextrose Agar (PDA) dengan menggunakan teknik monospora. Semua isolat ini kemudian diamati secara makroskopis dan mikroskopis untuk mengetahui morfologi koloni, pertumbuhan koloni, bentuk makrokonidium dan mikrokonidium serta konidiofornya. Hasil penelitian menunjukkan bahwa sebagian besar isolat mempunyai morfologi koloni yang hampir sama, sehingga tidak bisa dibedakan dengan jelas satu dengan lainnya. Akan tetapi, pengamatan secara mikroskopis menunjukkan adanya perbedaan morfologi konidiofor pada isolat F. oxysporum dengan Fusarium spp., sementara morfologi mikroskopis antara isolat Fusarium spp. sulit untuk dibedakan. Mikrokonidium yang dibentuk pada false-head merupakan karakter utama F. oxysporum. Dari hasil penelitian ini dapat disimpulkan bahwa morfologi mikroskopis hanya dapat digunakan untuk membedakan Fusarium spp pada tingkat spesies, tetapi tidak pada tingkat forma spesialis.