A new disease in Jatropha curcas L. plants was observed

during the summer season in 2009 and 2010 in

plantations in several Indian states, including Andhra

Pradesh, Assam, Chhattisgarh and Madhya Pradesh.

The outbreak of the disease coincided with the leafdropping

(dormant) stage of the crop, which sets in

with the increasing moisture stress. Affected J. curcas

plants showed drying along with shrivelling, and discoloration

of the stem with sticky reddish-brown exudation

at the base of the plants. Black lesions (soft and

rotting) on the stem under the bark and cambium

layer were also observed. From the affected stem

parts, fungal cultures were isolated and tested for

their pathogenicity on pot-grown plants. The fungal

isolates caused symptoms on J. curcas leaves and petioles

(black spots, 1–3 mm in diameter). Then they

spread to the stems causing shrivelling and gummosis

of hard-wood stems, and finally led to the death of the

infected plant. The causal fungus was identified as

Botryosphaeria dothidea using microscopic observations

of hyphae and spores, and internally transcribed

spacers (ITS) sequencing technique. In addition, four

other fungal isolates were also isolated from the affected

tissues, which were identified as Macrophomina phaceolina,

Phomosis longicolla, Fusarium oxysporum and

Alternaria alternata using the ITS sequencing technique.

The role of these fungal cultures, i.e. whether

they grow as saprophytes on the affected dead tissues

or have any role in causing the black rot disease, needs

further study. Spraying J. curcas plants showing early

symptoms of this disease with Bavistin (carbendazim

50% WP) at the rate of 2 g l–1 water controlled the

spread of the symptoms and led to the recovery of

plants with new leaf growth after the rains

Marimuthu, S

Pavani Kumari, M

Srinivasa Rao, Ch

Wani, S P

ICRISAT Open Access Repository

Occurrence of black rot in Jatropha

curcas L. plantations in India caused

by Botryosphaeria dothidea

RESEARCH COMMUNICATIONS  CURRENT SCIENCE, VOL. 100, NO. 10, 25 MAY 2011 1547*For correspondence. (e-mail: s.wani@cgiar.org) Occurrence of black rot in Jatropha curcas L. plantations in India caused by Botryosphaeria dothidea  Ch. Srinivasa Rao, M. Pavani Kumari,  Suhas P. Wani* and S. Marimuthu International Crops Research Institute for the Semi-Arid Tropics, Patancheru 502 324, India  A new disease in Jatropha curcas L. plants was obser-ved during the summer season in 2009 and 2010 in plantations in several Indian states, including Andhra Pradesh, Assam, Chhattisgarh and Madhya Pradesh. The outbreak of the disease coincided with the leaf-dropping (dormant) stage of the crop, which sets in with the increasing moisture stress. Affected J. curcas plants showed drying along with shrivelling, and dis-coloration of the stem with sticky reddish-brown exu-dation at the base of the plants. Black lesions (soft and rotting) on the stem under the bark and cambium layer were also observed. From the affected stem parts, fungal cultures were isolated and tested for their pathogenicity on pot-grown plants. The fungal isolates caused symptoms on J. curcas leaves and peti-oles (black spots, 1–3 mm in diameter). Then they spread to the stems causing shrivelling and gummosis of hard-wood stems, and finally led to the death of the infected plant. The causal fungus was identified as Botryosphaeria dothidea using microscopic observa-tions of hyphae and spores, and internally transcribed spacers (ITS) sequencing technique. In addition, four other fungal isolates were also isolated from the affected tissues, which were identified as Macrophomina pha-ceolina, Phomosis longicolla, Fusarium oxysporum and Alternaria alternata using the ITS sequencing tech-nique. The role of these fungal cultures, i.e. whether they grow as saprophytes on the affected dead tissues or have any role in causing the black rot disease, needs further study. Spraying J. curcas plants showing early symptoms of this disease with Bavistin (carbendazim 50% WP) at the rate of 2 g l–1 water controlled the spread of the symptoms and led to the recovery of plants with new leaf growth after the rains.  Keywords: Black rot, Botryosphaeria dothidea, drought-affected plants, dry season, Jatropha curcas.  JATROPHA CURCAS L. (physic nut), a large shrub reaching to a height of 5 m, belongs to the family Euphorbiaceae1. Although native to Central America, J. curcas is widely distributed in tropical and sub-tropical regions of Africa and Asia2,3. J. curcas is drought-tolerant, having a unique adaptation mechanism for moisture-stressed situations, during which it sheds all leaves to avoid transpiration and remains dormant – and then gives rise to new flushes once the moisture is recouped in the soil. The seeds of J. curcas contain 27–40% non-edible oil4. Due to the pres-ence of toxic components like phorbol esters and diterpe-noids, the Jatropha oil is non-edible5–8. In recent years, an increased interest in the cultivation of Jatropha as a source of biodiesel in several countries is being obser-ved9,10.  Jatropha is considered as a wonder plant due to its drought tolerance, unpalatability to animals and high oil content in its seeds. However, not much is known about its agronomic yield potential, agronomic practices, pests and disease occurrence and management11. The Interna-tional Crops Research Institute for the Semi-Arid Tropics (ICRISAT) has been exploring the use of Jatropha for rehabilitating the marginal lands and has planted block plantations in Velchal and Kothlapur villages in the Ranga Reddy District, Andhra Pradesh, India and also in its research farm at the ICRISAT centre in Patancheru (17°36′N, 78°16′E; 545 m altitude)4. A large-scale morta-lity of 3–4-yr-old Jatropha plants in Ranga Reddy and Kurnool districts, Andhra Pradesh (Figure 1) was obser-ved during June 2009 and 2010, which was initially thought to be due to the failure of the pre-monsoon rains during March–April 2009. Thirty eight per cent mortality of plants was recorded in a 140 ha block plantation in the Velchal village. In another 76 ha block plantation at Rol-lapadu and Cherukucherla villages in Midthur Mandal, Kurnool District, 80% of the plants were affected and whole plantation was lost. Close examination of the dead plants showed the symptoms of shrivelling and discolora-tion of stems at the base of the plants. Black lesions (rot-ting) under the bark and cambium regions of the stem (Figure 2) were observed, confirming the occurrence of disease, besides the impact of the drought. During April–May 2010, the affected Jatropha plants showed reddish-brown gummy exudation from the infected (shrivelling)      Figure 1. Severe mortality of Jatropha curcas occurred due to infec-tion caused by Botryosphaeria dothidea in Rollapadu village, Kurnool District, Andhra Pradesh, India, in August 2009. RESEARCH COMMUNICATIONS  CURRENT SCIENCE, VOL. 100, NO. 10, 25 MAY 2011 1548 parts of the stem, leading to the death of the plant (Figure 3). Occurrence and spread of this disease was mostly  observed in summer months, when there was a severe  water stress and drought conditions prevailed. There have been similar reports of the occurrence of such symptoms in commercial Jatropha plantations in Assam, Chhattis-garh and Madhya Pradesh (M. N. Pawar, pers. commun.), and affected samples were also used for this study.  The cultures isolated from the stems of the plants showing symptoms of rot were used for identifying the potential pathogens associated with the affected areas. Microscopic examination of the affected plant tissues  revealed the presence of scattered globose pycnidia near the margins of the black lesions. Conidia were ovulate, hyaline, aseptate, mostly single-celled and measured  20–32 × 12–15 μm. Bi-celled, brown, mature conidia were also observed (Figure 4). The organism was identi-fied as B. dothidea based on the characteristics of the  mature, bi-celled conidia and morphological observations using microscopy.   Disease-affected plant parts were collected from plan-tations in Andhra Pradesh and Chhattisgarh. Samples     Figure 2. Shrivelled appearance of black rot-affected stem (left) and black lesions over the stem under the bark (right) caused by B. do-thidea.     Figure 3. Exudation of reddish-brown gummy substance from the late infected stem (left) and discoloration of the affected stem (right) of J. curcas plants infected by B. dothidea. were cut into small pieces (~1 cm) and surface disin-fected with 5% sodium hypochlorite solution for 1 min, rinsed thoroughly with sterile distilled water and cultured on potato dextrose agar (PDA) plates. The plates were in-cubated at 25°C and after 4–5 days colonies developed on the affected stem pieces. The isolate was identified as B. dothidea on the basis of the colony morphology in vitro on PDA12 and hyphae characteristics studied using  microscopy and confirmed by ITS sequencing using the universal fungal primers ITS1 (5′-TCCGTAGGTGAAC-CTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATAT-GC-3′). The PCR product was sequenced at Macrogen Inc., Seoul, South Korea. Colonies on PDA were grey to black in colour with regular margin immersed along with light grey aerial mycelium, and the mycelium was hyaline and septate. B. dothidea is a known pathogen causing black rot in many other plants, including tree species13–15.  For proving pathogenicity, the standard Koch’s postu-lates process was adopted. Four centimetre long pieces of healthy stems were taken and the top epidermal layer was scraped using a scalpel and inoculated with 0.05–0.2 ml B. dothidea spore suspension (106 CFU/ml concentration) and tested for pathogenicity. The inoculated stems were covered with plastic bags and incubated at 25°C with a     Figure 4. Microscopic view of mature, bi-celled, brown conidia of  B. dothidea.     Figure 5. Developing light yellowish lesions in healthy leaves of Jat-ropha plant inoculated with spore suspension of B. dothidea after eight days of incubation. RESEARCH COMMUNICATIONS  CURRENT SCIENCE, VOL. 100, NO. 10, 25 MAY 2011 1549Table 1. Most potential fungal isolates from diseased J. curcas plant parts by BLAST analysis in ITS region    sequence analysis Code name      Organism Acc. No Identities (%)  JF01 Macrophomina phaseolina isolate MP112 GU046903.1  98 JF02 Phomopsis longicolla strain SM9713 FJ009527.1  97 JF03 Fusarium oxysporum isolate 158CH/S GU066709.1 100 JF04 Alternaria alternata isolate VC38 GQ169766.1 100 JF05 Botryosphaeria dothidea AY626347.1  98  relative humidity of 80%. After 48 h of incubation, the stem pieces were transferred to petri dishes. The colonies were observed around the inoculated area of the stem pieces on the third day of incubation. The colonies were white in colour initially and then turned black on the fifth day of incubation. The bark around the inoculated area shrivelled and turned black on the eighth day of incuba-tion. We also studied the pathogenicity by rubbing the in-fected (with spore suspension culture) stem pieces over the stems of healthy plants. Brown lesions were observed on leaves after eight days of incubation. The size of the  lesions ranged from 1 to 3 mm in diameter and similar  lesions were also noticed on the leaf petiole (Figure 5). The size of the lesions was enlarged in the infected leaf petiole on the 13th day of incubation. In another test, the entire plant was sprayed with a spore suspension (106 CFU/ml) and then covered with plastic bags to main-tain humidity during incubation in a glasshouse. How-ever, no symptoms were observed in the plants which were sprayed with the spore suspension in this manner. Koch’s postulates were completed by isolating and identi-fying the culture from artificially infected Jatropha plants, and hence the culture was confirmed as B. dothidea. For controlling the disease, the affected plant parts were pruned-off and sprayed with the fungicide Bavistin (car-bendazim 50% WP) at the rate of 2 g/l of water. After spraying the fungicide the recovery symptoms, i.e. growth of new sprouts from ground part of the stem and new foliage on the affected branches were observed.  Even though five pathogenic fungi were isolated from the disease-affected plant tissue, our laboratory experi-ment revealed that the symptoms that appeared on artifi-cially inoculated Jatropha plants with B. dothidea culture were similar to those in plants affected in the fields, and hence it was concluded that B. dothidea is the main causative agent of mortality in Jatropha plants. To our knowledge, this is the first report of the pathogen B.  dothidea causing black rot and severe damage to Jatro-pha in block plantations.    1. Divakara, B. N., Upadhyaya, H. D., Wani, S. P. and Laxmipathi Gowda, C. L., Biology and genetic improvement of J. curcas L., a review. Appl. Energy, 2010, 87, 732–742. 2. Wani, S. P., Osman, M., Emmanuel, D’Silva and Sreedevi, T. K., Improved livelihoods and environmental protection through bio-fuel plantation in Asia. Asian Biotechnol. Dev. Rev., 2006, 8, 11–29. 3. GEXSI LLP, Global Market Study on J. curcas, Final Report – Abstract. Prepared for the World Wide Fund for Nature, The Global Exchange for Social Investment, London/Berlin, 8 May 2008. 4. Wani, S. P., Sreedevi, T. K., Marimithu, S., Kesava Rao, A. V. R. and Vineela, C., Harnessing potential of J. curcas plantations for improving livelihoods and rehabilitating degraded lands. In Pro-ceedings of 6th International Biofuels Conference, Winrock Inter-national, New Delhi, 2009, pp. 256–273. 5. Makkar, H. P. S., Backer, K., Sporer, F. and Wink, M., Studies on nutritive potential and toxic constituents of different provenances of J. curcas. J. Agri. Food Chem., 1997, 45, 3152–3157. 6. Adolf, W., Opferkuch, H. J. and Hecker, E., Irritant phorbol de-rivatives from four J. curcas species. Phytochemistry, 1984, 23(1), 129–132. 7. Rakdhit, K. D., Darukeshwara, J., Raj, K. R., Narasimhamurthy, K., Saibaba, P. and Bhagya, S., Toxicity studies of detoxified J. curcas meal (J. curcas) in rats. Food Chem. Toxicol., 2008, 46, 3621–3625.  8. King, J. A., Wei, He., Cuevas, J. A., Ramiaramanana, D. and Gra-ham, I. A., Potential of J. curcas curcas as a source of renewable oil and animal feed. J. Exp. Bot. Rev., 2009, 60(10), 2897–2905. 9. Becker, K., Francis, G. and Edinger, R., A concept for simultane-ous wasteland reclamation, fuel production and socio-economic development in degraded lands in India: need, potential and per-spectives of J. curcas plantations, biodiesel from J. curcas planta-tions. Nat. Resour. Forum, 2005, 29, 2–24. 10. Wani, S. P., Sahrawat, K. L., Sreedevi, T. K., Bhattacharyya, T. and Sreenivasa Rao, Ch., Carbon sequestration in the semi-arid tropics for improving livelihoods. Int. J. Environ. Stud., 2007, 64, 719–727.  11. FAO, J. curcas: a small holder bioenergy crop. The potential for pro-poor development. Food and Agriculture Organization of the United Nations, Rome. Integrated Crop Management, 2010, 8, 114. 12. Boerema, G. H., De Gruyter, J., Noordeloos, M. E. and Hamers, M. E. C., Phoma Identification Manual. Differentiation of Specific and Infraspecific Taxa in Culture, CABI Publishing, UK, 2004. 13. Boerema, G. H., Contributions towards a monograph of Phoma (Celomycetes) – II. Section Peyronellae. Persoonia, 1993, 15, 197–221. 14. Blechtova, A., Ability of some micromycetes to cause necroses of apple tree bark in vitro. Ochrana Rostlin, 1995, 31, 265–276. 15. Pinter, C., Fischl, G., Kadlicsko, S., Danko, J., Gara, M. and Mako, S., Pathogens of walnut in Hungary. In Proceedings of the 52nd International Symposium on Crop Protection, Gent, Bel-gium, 9 May 2000, 65, 673–676.   ACKNOWLEDGEMENTS. We thank the National Oil Seeds and Vegetable Oils Development Board, Government of India, and the Government of Andhra Pradesh, Rain Shadow Area Development De-partment for financial support. We also thank Dr S. Gopalakrishnan for his suggestions and comments on this manuscript.  Received 11 October 2010; accepted 24 March 2011 

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Occurrence of black rot in Jatropha curcas L. plantations in India caused by Botryosphaeria dothidea

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