OCCURRENCE OF LEAF AND POD DISEASES OF SISSOO (DALBERGIA SISSOO ROXB.) IN BANGLADESH Shamim Shamsi1, Razia Sultana2 aana Azad3 1 Professor, 2M.Phil Student and 3MS Student, Department of Botany, University of Dhaka-1000, Bangladesh. Email: prof.shamsi@gmail.com ABSTRACT Shamim Shamsi, Razia Sultana and Rumana Azad. 2012. Occurrence of leaf and pod diseases of sissoo (Dalbergia sissoo Roxb.) In Bangladesh. Bangladesh J. Plant Pathol. Vol. 28(1&2): 33-40. sp., Lasiodiplodia theobromae (Pat.) Griffon and Maubol, Memnoniella sp., Phyllactinia dalbergiae Piroz. and its anamorph Ovulariopsis sissoo Shamsi, Sultana and Azad sp. nov, Tetraploa sp. and a rust fungus. From diseased pods, Colletotrichum gloeosporioides was isolated. This is the first report of association of Chalara sp., Memnoniella sp. and Tetraploa sp. with Sissoo from Bangladesh. A study was conducted to identify fungi associated with diseased leaves and pods of sisso tree (Dalbergia. sissoo Roxb (Sisam). Associated fungi were isolated and identified following standard methods. The associated fungi with leaves were Alternaria alternata (Fries) Keissler, Pseudocercospora dalbergiae (Sun) Yen, Chalara sp., Colletotrichum gloeosporioides (Penz.) Sacc., Cylindrocladium sp., Fusarium solani (Mort.) Sacc., Gebberella Keywords: Mycoflora, association, leaves and pods, Dalbergia sissoo Lasiodiplodia theobromae from dieback symptom of sissoo. Shamsi et al. (2008) reported Phyllactinia dalbergiae and Ovulariopsis sissoo from powdery mildew infected plant parts. Information about fungi associated with diseased leves and pods of Sisso tree is limited in Bangladesh (Shamsi et al. 2008). The present study was conducted to identify fungi associated with diseased leaves and pods of Sisso trees. INTRODUCTION Dalbergia sissoo Roxb. (Sisam) is an important timber tree with high timber value. The plants are attacked by a number of diseases such as powdery mildew, leaf rust, leaf blight, collar rot, wilt, die-back and Ganoderma root rot of the tree are reported by various researchers. The plant is susceptible to dieback, wilt and several other soil borne pathogens (Sah et al. 2003). Mukerji and Bhasin (1986) reported leaf spots caused by Cercospora sissoo, Cochliobolus lunatus, Colletotrichum sissoo, Cylindrocladium scoparium, Phomopsis dalbergiae, Phyllachora spissa, Phyllosticta sissoo; leaf blight caused by Colletotrichum gloeosporioides; leaf rust caused by Maravalia achora, Uredo sissoo and powdery mildew caused by Phyllactinia dalbergiae from India. Pod was infected by Catenulaster batistae, Glomerella cingulata and Septothyrella dalbergiae. Bakshi (1974) isolated Phellinus gilvus from roots of trees affected by dieback. Richardson (1990) reported several species of Aspergillus, Penicillium, Rhizopus, Alternaria, Fusarium, Chaetomium, Drechslera and Curvularia from forest tree seeds. Parajuli et al. (1999) reported Fusarium oxysporum from Dalbergia sissoo on water-logged soils in Nepal. Manadhar et al. (2000) isolated Botryodiplodia sp. and Fusarium solani from five diseased samples of D. sissoo. Khan et al. (2001) detected Aspergillus niger, A. flavus, A. terreus, Aspergillus sp., Alternaria alternata, Chaetomium sp., Drechslera australiensis, Fusarium pallidoroseum, F. solani, Fusarium sp., Penicillium sp., Rhizopus sp., and Geotrichum sp. from seeds of shisham trees. Rajput et al. (2008) isolated F. solani, Rhizoctonia solani and Curvularia lunata as predominant fungi from shisham trees infected with dieback. From Bangladesh, Muehback et al. (2010) isolated F. oxysporum and MATERIALS AND METHODS Diseased leaf and pod samples of Sisso were collected from Dhaka, Chittagong and Pabna districts during October 2008 to January 2010. Associated symptoms were recorded. The severity of disease on leaves was estimated visually using a 0-9 subjective scale, where 0= no infection, 1= up to 10% leaf area infected, 2= 10 – 20% leaf area infected, 3=20 – 30% leaf area infected, 4= 30 – 40% leaf area infected, 5= 40 – 50% leaf area infected, 6= 50 – 60% leaf area infected, 7= 60 – 70% leaf area infected 8= 70 – 80% leaf area infected and 9= 80% and above leaf area infected (Ghos et al. 2009). Fungi associated with diseased samples were isolated following “tissue planting method" and "blotter method” (Anon. 1968). In case of “tissue planting method” fifty inocula each measuring 2 mm2 was cut from a particular specimen. The inocula were washed in sterile water and surface sterilized by dipping in 10.0% Clorox for 3-5 minutes. Three inocula were placed in each Petri plate containing sterilized potato dextrose agar (PDA) medium and incubated for 5-7 days at 25±2C. In “blotter method” moist chambers were made by placing two layers of filter paper on the bottom of the Petri plates. In each Petri plate, 5 surface sterilized inocula were placed and 10 plates were used. The inoculated plates 33 were incubated at room temperature. The fungi growing out of the inocula were transferred to separate PDA plates and slants, and stored in a refrigerator for further studies. Prevalence of fungi associated with the specimens was expressed in percentage based on total number of leaf and pod samples checked. Identification of the isolated fungi was done using standard literature (Booth 1971, Ellis 1971, 1976, Ellis and Ellis 1982, Sutton 1980). All the specimens were preserved in the Herbarium, Mycology and Plant Pathology Section, Department of Botany, University of Dhaka, Bangladesh. Leaf spot: The disease appears as small circular spots on leaves. The size of the spots increases with the progress of times. Larger spots develop due to coalescence of closure spots (Plate IV C). The causal pathogen, Pseudocercospora dalbergiae (Plate IV D) attacks the leaves mostly on the lower surface of leaves producing yellowish to grayish-green discoloration. The mycelium is brown and intra-epidermal. The fungus produces asexual fruiting structure in July and August 2009. In the month of January, its severity index was 4. The highest disease index of 7 was recorded in the month of October and 5 in the month of November 2009 (Fig. 1 and 2 and Table 1). RESULTS AND DISCUSSION Leaf blight: The leaf blight symptoms was associated with leaf samples collected from Savar (Plate IV A and Table 1). Several fungi namely Alternaria alternata, Chalara sp., Cylindrocladium sp., Fusarium solani, Lasiodiplodia theobromae, Memnoniella sp. and Tetraploa sp. were found to be associated with leaf blight infected samples (Plate V B-H). The fungi were associated with leaf samples during June to October 2009 and their prevalence ranged 5.88-16.67% (Fig. 1). Different types of symptoms representing seven diseases were found on diseased leaves of D. sissoo. These were anthracnose, powdery mildew, angular leaf spot, leaf blight, leaf spot and leaf rust. Anthracnose symptom was also noticed on infected pods (Table 1 and Plate I-VI). Their severity in different months are shown in Figure 1. Anthracnose: Anthracnose was found in all leaf samples (Plate I A) collected from Savar and Pabna. The disease also appeared on pod samples (Plate I B & C) collected from only Pabna (Table 1). The highest disease severity index of 8 was recorded in the month of March and the index value was 6 during April to December 2009 (Fig. 1). Colletotrichum gloeosporioides was associated with leaf and pod samples infected with anthracnose (Plate I D). Rust: The rust disease appeared during January to March on leaves (Plate VI) and young twigs. Uredinial sori are yellowish and formed on the lower surface of the leaves (Plate V). The fungus was found in the samples collected from Savar (Table 1) It severity varied with months (Fig. 1). Present report is slightly differing from the observation of Bakshi (1967). The author reported that the rust disease appeared in January to March on leaves and juvenile twigs and continued attacking the foliage and young twigs up to July and August. The infection declines following monsoon rains. The affected parts are killed resulting in die-back and subsequent death of affected seedlings. Maravalia achroa is recorded on seedling in nurseries from Uttar Pradesh, Bihar, Maharashtra and Assam. The disease also occurs on young plantations but not in as severe form as in the nurseries. Powdery mildew: Symptoms of powdery mildew caused by imperfect stage of the pathogen (Ovulariopsis sissoo Shamsi, Sultana and Azad) (Plate II A & B) were recorded from five leaf samples collected from Dhaka (Table 1). Powdery mildew symptoms developed by perfect stage of the fungus (Phyllactinia dalbergiae) (Plate III A & B) were found on two leaf samples collected from Dhaka and two from Savar (Table 1). The infection starts in the middle of October 2008 with the formation of a white mycelial growth mostly on the lower surface of the leaves. With the age, the mycelium and conidiophores bearing conidia become grayish-white to pale yellow. By the end of December most of the leaves on the trees are covered with white colony growth of the fungus, perithecia start to form, which were initially orange in colour. The ascocarps turn into brown with the progress of the disease and ultimately become black at maturity. The powdery mildew causes severe defoliation but never kills the tree. New leaves develop with the advent of spring. The highest severity index of 8 was recorded in the month of January and 7 in November and December, 2009. Perithecia formed during February to March. Perithecia were found in the samples collected from Dhaka (Fig. 1 and Table 1). Fungi associated with diseased leaves and fruits: A total of 13 fungal species namely, Alternaria alternata, Pseudocercospora dalbergiae, Chalara sp., Colletotrichum gloeosporioides, Cylindrocladium sp., Fusarium solani, Gibberella sp., Lasiodiplodia theobromae, Memnoniella sp., Ovulariopsis sissoo, Phyllactinia dalbergiae, Tetraploa sp. and an unidentified rust fungus were found to be associated with infected leaves of D. sissoo. Prevalenc of P. dalbergiae was the highest followed by C. gloeosporioides, O. sissoo, and A. alternata showing the prevalence of 52.94, 46.67 and 16.67%. Prevalence of F solani, P. dalbergiae and Gibberella sp. was 11.76, 11.76 and 11.11%, respectively. The most prevalence of 5.88 was recorded in Chalara sp., Cylindrocladium sp., Memnoniella sp., rust fungus and Tetraploa sp. Colletotrichum gloeosporioides was isolated from infected fruits (Fig. 2). Angular leaf spot: Angular leaf spot symptoms (Plate IV A) were frequently noticed on leaves collected from Chittagong (Table 1) was found in the samples. The causal fungus of angular leaf spot was identified as Gibberella sp. (Plate IV B). 34 Table 1. Fungi associated with leaf and pod samples of Dalbergia sissoo having different types of symptoms collected from various locations of Bangladesh during December 2008 to January 2010 Date of collention 08-12-2008 27-01-2009 10-03-2009 17-06-2009 22-07-2009 04-07-2009 22-01-2009 17-02-2009 15-11-2009 06-12-2009 19-02-2009 02-03-2009 05-03-2009 SS 2184 22-11-2009 SS 2172 08-07-2009 SS 2170 SS 2175 SS 2177 SS 2179 SS 2180 SS 2182 SS 2181 SS 2186 SS 2207 17-06-2009 22-07-2009 05-08-2009 19-08-2009 26-08-2009 07-10-2009 23-11-2009 12-12-2009 12-01-2010 Locality Pabna Pabna Savar Savar Savar Savar Dhaka Dhaka Dhaka Dhaka Dhaka Dhaka Dhaka Diseases Anthracnose Anthracnose Anthracnose Anthracnose Anthracnose Anthracnose Anthracnose Powdery mildew Powdery mildew Powdery mildew Powdery mildew Powdery mildew Powdery mildiew with perithicia Savar Powdery mildiew with perithicia Savar Powdery mildiew with perithicia Savar Blight Savar Blight Savar Blight Savar Blight Savar Blight Savar Blight Chittagong Angular spot Savar Rust Savar Rust Plant parts Leaf Pod Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Identified fungi Colletotrichum gloeosporioides Colletotrichum gloeosporioides Colletotrichum gloeosporioides Colletotrichum gloeosporioides Colletotrichum gloeosporioides Colletotrichum gloeosporioides Ovulariopsis sissoo Ovulariopsis sissoo Ovulariopsis sissoo Ovulariopsis sissoo Ovulariopsis sissoo P. dalbergiae Phyllactinia dalbergiae Leaf P. dalbergiae Leaf P. dalbergiae Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Leaf Fusarium solani Alternaria alternata Tetraploa sp. L. theobromae Memnoniella sp. Cylindrocladium sp. Gibberella sp. Urediospor Urediospor Disease severity (0-9) Sample No. SS 2136 SS 2138 SS 2164 SS 2171 SS 2174 SS 2184 SS 2152 SS 2157 SS 2183 SS 2185 SS 2158 SS 2160 SS 2163 Fig. 1. Disease progress curve of powdery mildew (PM), anthracnose (AN) and leaf spot (LS) symptoms recorded on Dalbergia sissoo from January to December 2009. 35 B A D C Plate I. Photographs showing symptoms of anthracnose on leaf (A) an pods (B&C) of Dalbergia sissoo caused by Colletotrichum gloeosporioides (D) B A Plate II. Photographs showing symptoms of powdery mildew on leaf of Dalbergia sissoo caused by Ovulariopsis sissoo ((A & B) [Bar = 50 µm]. 36 B A Plate III. Photographs showing symptoms of powdery mildew on leaf of Dalbergia sissoo caused by Phyllactinia dalbergiae (A & B) [Bar = 50 µm]. B A D C Plate IV. Photographs showing symptoms of angular leaf spot (A) caused by Gibberella sp. (B) and leaf spot (C) caused by Pseudocercospora dalbergiae (D). 37 C A B Leaf blight symptoms Alternaria alternata Chalara sp. F E D Cylindrocladium sp. Fusarium solani G Lasiodiplodia theobromae. H Memnoniella sp. Tetraploa sp. Plate V. Photographs showing symptoms of leaf blight symptoms (A) of associated with the blighted leaves(B-H) (Bar = 50 µm) 38 Dalbergia sissoo and fungi B A Rust disease An unidentified species of rust fungus Plate VI. Symptoms of rust disease on leaf (A) of Dalbergia sissoo caused by an unknown species of rust fungus (B) (Bar = 50 µm) 70 60 Prevalence (%) 50 40 30 52.94 46.67 20 29.41 10 16.67 11.76 11.11 5.88 5.88 5.88 5.88 5.88 11.76 5.88 ia e es o C Ps . eo s da l po rio be rg id .s is so O at a ltr n A. a al be rg ia e .g lo G P. d ib be re l F. so la ni sp . la sp . pl oa ra Te t tf un gu s sp . el la no ni em M R us ae om eo br th L. ro cl a C yl in d C ha la ra di um sp . sp . 0 Fungi Fig. 1. 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