1. Sajad un Nabi 1 , Madhupriya 1 and G.P.Rao 1
Hishimonus phycitis (Distant), a potential vector for sesame phyllody phytoplasma in India
Sajad un Nabi 1 , Madhupriya 1 and G.P.Rao 1
1Division of Plant Pathology , Indian Agricultural Research Institute, New Delhi
Presenting author
ABSTRACT
Phyllody symptoms were observed on sesame plants during September-October 2013, in sesame fields at Indian Agricultural Research Institute (IARI), New Delhi with incidence of 20-30%. Three different leafhopper species viz. Empoasca prima (EP), Hishimonus phycitis (HP) and Exitinus indicus (EI) were found as the most dominant feeding species at different stages of crop growth. The population dynamics studied shows HP as the most predominant species at the time of symptoms initiation in sesame crop and it’s population was also correlated with increase in sesame phyllody (SP) incidence. DNA was isolated from all the three leafhopper species and symptomatic sesame plants. Phytoplasma association was confirmed with symptomatic sesame plants utilizing P1/P6 and nested primer pair RI6F2n/RI6R2n which yielded exclusive bands of ~1.5kb and ~1.25kb, respectively. However, ~1.25kb product was amplified only with HP in nested PCR assays. No amplification was observed from either of the other two leafhopper species (EP,EI) or healthy sesame plants. The 1.25kb product was directly sequenced and BLAST analysis of 16SrDNA sequence from HP and SP infected plants revealed 100% sequence similarity between themselves and 99% with phytoplasma members of the group ‘Ca.P. asteris’ (16Sr I) group. Phylogenetic analysis further confirmed the closest phylogenetic relationship of the SP and HP isolate with those of ‘Ca.P.asteris group’ (16Sr I).The virtual RFLP pattern of the SP and HP isolates in the present study suggested the association of subgroup B (16SrI-B) which was further confirmed through actual RFLP using different restriction endonucleases(BamHI,BfaI,EcoRI,HhaI,HpaII,RsaI,) with 1.25kb nested PCR product. The identity of sesame phyllody phytoplasma in HP suggested that Hishimonus phycitis may act as potential vector for sesame phyllody phytoplasma transmission in nature
( a) (b) (c)
Fig 1: Sesame plants(a) Healthy (b) witches broom (c) phyllody
Fig 3: Population dynamics of EI,HP,EP in relation to sesame phyllody incidence
Fig 2: Leaf hopper (Hishimonus phycitis)
INTRODUCTION
Phytoplasmas are phytopathogenic prokaryotes without cell wall and inhabit phloem sieve elements in infected plants belongs to class Mollicutes.
Genome of phytoplasmas is the smallest among known cellular plant parasites and pathogens of size 600 to 1600 kbp, present as one large circular double stranded DNA chromosome.
Sesame phyllody caused by phytoplasma is one of the major constraints throughout the world as well as in India, which causes losses of 5-15%.
The phytoplasma is transmitted from infected to healthy plants by phloem-feeding insects, mainly leafhoppers belongs to order Hemiptera.
Orosius albicinctus, Neoaliturus haematoceps Circulifer haematoceps leafhopper vector for sesame has been reported as potential vector of sesame phyllody phytoplasma in nature in India and abroad.
Survey: During september 2013 survey was done in IARI sesame fields to collect sesame phyllody plants and leafhoppers
Population dynamics of Leafhoppers: Leafhoppers were collected by sweeping net and population dynamics was studied using yellow insect traps at ten days interval.
DNA isolation: DNA was extracted separately from 20 individuals of each leafhopper species and the affected sesame phyllody (SP) plant by Cetyl Trimethyl ammonium bromide (CTAB) method(Ahrens and Seemuller, 1992).
PCR assays: Primer pair P1/P6 (Deng and Hiruki, 1991) was used for first round PCR and R16F2n/R16R2(Gundersen and Lee, 1996) for nested PCR.
Sequencing and alignment: 1.25 kb amplicon was purified and sequenced directly in both directions. The sequence generated was aligned using CLUSTAL W method of Bio-Edit software (Bio-edit Sequence Align Editor).
Phylogeny analysis :The sequence generated from the present study and reference phytoplasma strains sequence retrieved from GenBank were used to construct phylogeny through MEGA 5.0 version software.
Virtual RFLP: iphy classifier available on USDA website ( to confirm the subgroup of the phytoplasma ,which was further confirmed through actual RFLP by using Restriction enzymes(BamHI,BfaI,EcoRI,HhaI,HpaII,RsaI).
Suspected sesame phyllody symptoms were observed where Inflorescence was converted into twisted reduced green leaves, with short internodes (witches broom) and transformation of floral parts into green leaf-like structures known as phyllody (Fig.1
Leafhoppers collected were identified as Exitianus indicus (Distant)(EI), Hishimonas phycitis (Distant) (HP) (Fig 2), and Empoasca prima (EP).Population dynamics revealed the direct correlation of SP with increase in population of Hishimonus phycitis (Fig 3)
PCR amplification of the 16S rRNA infected sesame plant sample yielded 1.5 kb product in the first round PCR with primer pair P1/P6. However, ~1.25kb product was amplified only with HP in nested PCR assays and no amplification was observed from either of the other two leafhopper species (EP,EI) or healthy looking sesame plants. (Fig. 4).
BLAST analysis of the 16S rRNA partial sequence of H. phycitis (Distant) phytoplasma isolate and the sesame phyllody revealed 100% similarity among themselves and 99% with ‘Ca P. asteris’ (16Sr I group).
Phylogenetic analysis further confirmed that the phytoplasma associated with the H. phycitis (Distant) belonged to ‘Ca.P.asteris’ group (16Sr I group) (Fig 5).
The virtual(Fig:6) and actual RFLP(Fig7) confirmed that both HP and SP isolates belong to subgroup B (16Sr I-B)
M SP HP EI EP P
1.25kb
Fig 4 : Nested PCR assay results of SP:Sesame phyllody plants, HP:Hishimonus phycitis,EP:
Empoasca prima, EI: Exitinus indicus, P:Positive control, M:Marker 1kb DNA ladder
Sesame phyllody Deorian-UP(KC920749)
Mulberry dwarf phytoplasma(AB693124)
Angustata ash witches-broom (AB693130)
Onion yellows (NR )
Aster yellows (HM467127)
Sesame phyllody Kushinagar(KC920747)
Rhus yellows (AB )
Hishimonus phycitis (KF826901)
Sesame phyllody IARI( KF744233)
Peanut witches broom (L33765)
Sugarcane white leaf (X76432)
Pigeon pea witches broom (AF248957)
Coconut lethal yellowing (AF498307)
Alder yellows (AY )
Fragaria Multicipita (AF036354)
Erigeron witches broom (AY034608)
A.Laidlawii (M23932 ) 52 91 43 39 98
100
0.01
experimentals
16Sr I
Fig 5: Phylogenetic relationship between SP and HP phytoplasma with reference strains with accession numbers in phylogenetic tree
M BamHI Bfa1 EcoR1 Hha1 Hpa11 Rsa1 M
Result and Discussion
Fig:6 Virtual RFLP pattern derived from Insilco digestion 1.25kb 16SrDNA sequence of SP Ca. phytoplasma using 17 different Restriction Endonucleases indicating that SP and HP belong to subgroup B (16SrI-B)
Fig 7:Actual RFLP analysis of 1.2kb product of SP and HP digested with RestrictionenzymesBamHI, BfaI, ,EcoRI,
HhaI,HpaII,RsaI,M:phi x 174/Hae III marker shows similar restriction sites as on virtual
CONCLUSION AND FUTURE THRUST
The population dynamics and detection of sesame phyllody phytoplasma in Hishimonus phycitis suggests that it may play significant role as vector for sesame phyllody phytoplasma transmission in nature.
Further studies need multilocus gene characterization and transmission tests to confirm association of leafhopper species Hishimonus phycitis as a vector for SP phytoplasma transmission in nature.
IMPORTANT REFERENCES
Ahrens,U.and SeemüllerE Phytopathology , 82:
Akhtar K,P. et al Turk J Agric For. 33;
Azadvar ,M and Baranwal,V.K (2012). Phytopathogenic Mollicutes 2 (1):15-21.
Gundersen, D. E. and Lee, I.-M. (1996).. Phytopathol Mediterraneae 35:
Deng, S. and Hiruki, C. (1991) Journal of Microbiological Methods, 14 :53-61.