Babu, M. Kochu

Jayanthi, M.

Mandal, P.K.

Satyavani, V.

English

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392Journal of Plantation Crops, 2014, 42(3):392-394Short Scientific ReportPCR based early detection of Ganoderma sp. causing basalstem rot of oil palm in IndiaP.K. Mandal*, M. Kochu Babu, M. Jayanthi, and V. SatyavaniDirectorate of Oil Palm Research, Pedavegi, West Godavari District, Andhra Pradesh, India(Manuscript Received: 22-08-13, Revised:26-07-14, Accepted: 22-09-14)Keywords: Ganoderma, PCR, diagnosis*Corresponding Author: pranabkumarmandal@gmail.comBasal stem rot (BSR) is one of the most seriousdiseases of oil palm and the incidence could be ashigh as 25 per cent (Singh, 1991; Ariffin et al.,2000).  In India, the disease is caused by eitherG. lucidum or G. applanatum (Anonymous, 2008).Cross infectivity of this pathogen is already reported(Turner, 1981; Mandal et al., 2003) and the BSRoccurrence in oil palm in India is noticed whereunder planting is done in the infected coconutgardens. The disease is rapidly spreading in the oilpalm growing states of India. One of the majorproblems associated with this disease managementis the lack of early diagnosis. By the time symptomappears, the palm is beyond recovery. If earlyidentification of infected palms was impossible,they can be treated and the disease can be managedeffectively. Hence an early detection technique isfelt essential for effective management of basal stemrot in oil palm.Ganoderma is a soil borne fungi under thefamily of Ganodermataceae, which mostly causeinfection through the root. Hence the inoculum canbe detected first in the root than from any otherpart of the palm. In the present study, a PCR basedconfirmatory diagnostic technology for detectionof Ganoderma has been attempted using the rootsamples of oil palm.To standardize the PCR protocol foridentification of Ganoderma, BSR affected palmswere located and infected tissues were collectedfrom the base of the stem. Ganoderma isolates werepurified in 2 per cent potato dextrose agar (PDA)medium in Petri plates. Mycelia (200 mg) from twoweeks old Ganoderma isolates were used for DNAextraction using the miniprep method using 1 mLpre-heated DNA extraction buffer containing50 mM Tris-HCl, 10 mM EDTA, 100 mM NaCland SDS 1 per cent.  Crude DNA preparation wastreated with RNase and phenol:chloroform (1:1) forpurification.Amplification of the DNA samples was carriedout using two sets of virulent Ganoderma specificprimers - Gan1 (5’-TTG ACT GGG TTG TAGCTG-3’) and Gan2 (5’-GCG TTA CAT CGC AATACA-3’) (Utomo and Neipold, 2000); and  Gan ET(5’-GAG TTG TCC CAA TAA C-3’) and ITS3(5’-GCA TCG ATG AAG AAC GCA GC-3’), whichwas derived from the ITS region of Ganoderma(Bridge et al., 2000). DNA (5 ng), dNTPs (50 mMeach), primers (5 ng each) and Taq (0.5 U)polymerase were mixed to a reaction volume of12.5 μL for amplification. PCR condition for boththe sets of primers was set as 95 OC - 5 min.;40 cycles of  94 OC- 35 sec., 52 OC- 35 sec.,72 OC- 40 sec.; finally 72 OC - 10 min for amplification.To the amplified product, 1.5 μL bromophenolblue dye was added and 10 μL sample waselectrophoresed in 2 per cent  agarose gel. Thegel was stained with ethidium bromide andviewed in a gel documentation system.All the Ganoderma isolates showedamplifications with both the primer pairs. Gan1 -Gan2 produced a 167 bp DNA fragment and GanET-ITS3 produced a 320 bp DNA fragment. However,393the 320 bp fragment was more prominent. To detectthe presence of infection, healthy looking rootsamples from infected palms were collected andDNA was extracted using the method mentionedabove as well as by CTAB method. DNApreparation was purified using RNase andphenol:chloroform (1:1) and subjected toamplification by PCR using the procedurementioned above. But no amplification could benoticed. The possible reason for non-amplificationmight be due to insignificant amount GanodermaDNA present in the DNA preparation in comparisonto the plant DNA.Thereafter, the healthy looking root samplesfrom infected palms were surface sterilized for1 min with 0.1 per cent mercuric chloride and cutin 1 cm pieces. The root pieces were cultured on2 per cent PDA media in Petri plates. They wereincubated at 28 OC temperature for 5 days andmycelial growth was visible on and around the rootpieces (Fig. 1). Visible mycelial growth was notprominent in all the cases; however, the purposewas to proliferate the organism present in the rootfor a period of 5 days. Surface of the root pieces,the surrounding surface area of the PDA media werescraped out gently along with mycelia (whereverpresent) with a sterile blade and around 50 mg ofmaterial was used for DNA extraction usingminiprep method described earlier. Both the primerswere used for DNA amplification and PCR productsrevealed the desired fragment of 167 bp and 320 bpby Gan1-Gan2 and GanET-ITS3 primersrespectively (Fig. 2). It is important to note that,when root samples were incubated for 3 days andfollowed the procedure, amplifications were noticedFig. 1. Root samples showing mycelia growth, five daysafter inoculationFig. 2. Amplification of Ganoderma samples using Gan1-Gan2  and GanET-ITS3 primers. Legend: Lane M:100 bp ladder; Lanes 1 to 10: 167 bp (top) and 320 bp(bottom) fragments produced by amplification of DNAextracted from the material surrounding root samples(healty looking root from BSR infected palms)  5 daysafter incubation in 2% PDA using Gan1 - Gan2 andGanET – ITS3 primers respectively. Lane 11: positivecontrol (DNA from confirmed Ganoderma isolates);Lane 12: Negative control.Fig. 3.  Amplification of random root samples using GanET-ITS3 primers. Legend: Lane M: 100 bp ladder; Lanes1 to 15: amplification of DNA extracted from randomlycollected root samples, 5 days after incubation in 2%PDA. Lane 16: negative control; Lane 17: positivecontrol.in most of the cases and in all the cases withprominence when incubated for 4 days. However,5 days incubation is recommended with anallowance of an additional day.Subsequently, 15 root samples were collectedrandomly from the healthy looking oil palms fromdifferent basal stem rot affected gardens located inWest and East Godavari districts of Andhra Pradeshand they were incubated for 5 days in 2 per centPDA medium as mentioned above. DNA from thosesamples was subjected to PCR amplification usingGanET-ITS3 primers (Fig. 3).  Five samples out offifteen showed the presence 320 kb fragment. Thesame samples were reconfirmed using Gan1- Gan2Early detection of Ganoderma in oil palm394primers also (Fig. 4). When root bits were incubatedfor 5 days in PDA media, the negative samples alsoshowed some mycelial growth. This might be dueto the presence of some other soil borne fungi, butdefinitely not Ganoderma as confirmed by PCR.Some progress on development of precisetechnique for early detection of Ganoderma werereported through enzyme-linked immunosorbentassay (ELISA) using polyclonal antibody and PCRtechniques elsewhere (Utomo and Neipold, 2000;Darmono and Suharyanto, 1995). In India, BSR inoil palm was reported only from Tripura state(Sankaran et al., 2005).  But the disease may beprevalent, which goes unrecorded, as sporocarps arenot produced during early stage of infection.However,  incidence of the disease  in oil palm wasnoticed during 2002 (Kochu Babu  and Mandal,2003) from Andhra Pradesh and Kerala.  Thereafter,it is increasing in alarming way, especially in theEast and West Godavari districts of Andhra Pradesh,which necessitates the need for an authentic earlydiagnostic tool. This report validates and confirmsthe PCR based early detection of Ganodermacausing basal stem rot disease in oil palm, which isaccurate and robust. The significance of the studylies on routine survey and random root samplescollection from different oil palm gardens. Earlydetection would be possible three to five days aftercollection of samples by using this methodology.Fig. 4. Confirmation of Ganoderma infection in the healthylooking root samples using GanET-ITS3  and Gan1-Gan2 primers. Legend: Lane M: 100 bp ladder; Lanes1 to 5: amplification of random root samples havingGanoderma infection using GanET – ITS3 primers;Lanes 6: positive control using GanET – ITS3 primers;Lanes 7 to 11: amplification of same samples (lane1-5) using Gan1 – Gan2 rimers; Lanes 12: positivecontrol using amplification using Gan1 – Gan2primers.Subsequently, appropriate control measures can betaken up to save the affected palms as well as spreadof the disease from one palm to other. Thus, this isan important step in managing BSR disease of oilpalm in the country.ReferencesAnonymous. 2008. Studies on diseases of oil palm and theirmanagement.  In: Annual Report 2007-08 NationalResearch Centre for Oil Palm,  Published by Director,National Research Centre for Oil Palm, Pedavegi, WestGodavari District, Andhra Pradesh. pp.58-60.Ariffin, D., Idris, A.S. and Singh, G. 2000. Status ofGanoderma in oil palm. In: Ganoderma Diseases ofPerennial Crops (Eds.) Folld, J., Bridge, P.D. andHolderness, M. CABI Publishing, CAB International,Wallingford, UK. pp. 49-68.Bridge, P.D., Grady, E.B.O., Plotti, C.A. and Sanderson, F.R.2000. Development of molecular diagnostics fordetection of Ganoderma isolates pathogenic to oil palm.In: Ganoderma Diseases of Perennial Crops (Eds.) Folld,J., Bridge, P.D. and Holderness, M. CABI Publishing,CAB International, Wallingford, UK. pp. 225-247.Darmono, T.W. and  Suharyanto, A.  1995. Recognition offield materials of Ganoderma sp. associated with basalstem rot in oil palm by a polyclonal antibody. MenaraPerkebunan 63: 15-22.Kochu Babu, M. and Mandal, P.K. 2003. In:  Annual Report2002-03 National Research Centre for Oil Palm,Pedavegi, West Godavari District, Andhra Pradesh, India,pp. 49-50.Mandal, P.K., Hymavathi, K., Jayanthi, M. and Kochu Babu,M. 2003. Cross infectivity study of Ganoderma lucidumfrom coconut to oil palm and their genetic diversity studyby RAPD and AFLP. International Journal of Oil Palm,3&4: 57-60.Sankaran, K.V., Bridge, P.D. and Gokulapalan, C. 2005.Ganoderma diseases of perennial crops in India - anoverview. Mycopathologia 159(1): 143-152.Singh, G. 1991. Ganoderma – the scourge of oil palm in thecoastal areas. The Planter  67: 421-444.Turner, P.D. 1981. Oil Palm Disease and Disorders inMalaysia. Incorporated Society of Planters, KualaLumpur, Malaysia. 247 p.Utomo, C. and  Niepold, F. 2000. Development of diagnosticmethods for detecting Ganoderma infected oil palms.Journal of  Phytopathology 148: 507-514.Mandal et al.

PCR based early detection of Ganoderma sp. causing basal stem rot of oil palm in India

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PCR based early detection of Ganoderma sp. causing basal stem rot of oil palm in India

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