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DEPARTMENT OF PLANT PATHOLOGY

Assam Agricultural University

Jorhat-785013, Assam

THESIS SEMINAR

TITLE: Identification and Characterization of Citrus Greening

Disease of Assam

Name: MAITRY PAUL Time: 11.00amRegd.No: 11-AMJ-78 Venue:Seminar HallMajor Advisor: Dr. P. D. NATHDate: 06.07.13

ABSTRACT

An attempt was made to identify and characterize the citrus greening disease (CGD) of Assam.

Survey was conducted in 8 districts viz. Jorhat, Tinsukia, Dibrugarh, Kamrup, North Lakhimpur,Nagaon, Cachar and North Cachar Hills comprising six Agro-climatic zones of Assam to

determine the presence and incidence of Citrus greening disease (CGD) based on polymerase chain

reaction (PCR) and their biological and molecular characterization. Samples were collected fromtwo important commercial species namely, Khasi mandarin (Citrus reticulata) and Assam lemon

(Citrus limon). Characteristic symptoms of mottled or blotchy yellow leaves on an individual

branch or only in one portion of the canopy of the tree were observed on CGD infected plants.Yellow shoot, severe chlorosis, scattered green spots, vein clearing and yellowing symptoms were

also observed. The fruits were under-developed, lopsided, and poorly colored with aborted seeds.Biological indexing recorded chlorosis and scattered greening symptoms on the indicator host,

sweet orange (Citrus sinensis) from 5th to 9th weeks after inoculation. Visual observation ofsymptom expression and PCR assay of indicator host recorded 40 per cent and 60 per cent

symptom expression, respectively at the end of 9 th week. PCR analysis was done to amplify the

specific DNA fragments from CGD infected and healthy samples. PCR results revealed that theCGD specific primer pair had yielded a product of 703 bp. The results revealed a total of 37

positive and 82 negative samples indicating 31.09 per cent CGD incidence in Assam. PCR

product of CGD infected samples were sequenced and compared with known CGD from India andabroad using nucleotide BLAST programme at National Centre for Bio Informatics (NCBI) and

MEGA 5.05 software. Sequence similarity analysis showed that CGD isolates of Assam have 100

% similarity with Umiam isolate, 99% similarity with Maharashtra, Iran, USA, Italy and China and80% similarity with South African isolate. This gave an indication that the CGD has similaritywith other CGD isolates already reported. Samples were then assayed using real time PCR against

multiplex primers and probes of C l asiaticus, C l americanus and C l africanus. The result

suggested the presence ofC l asiaticus and C l africanus strains. Hence the samples were furtherassayed using C l asiaticus and C l africanus specific primers and probes. The result recorded 100

% positive samples against C l asiaticus and C l africanus in all the 8 districts. This suggested the

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prevalence of dual infection ofCl asiaticus and C l africanus strains in all the 6 agroclimatic zones

of Assam.