Recent Biotechnological Approaches in Diagnosis and Management of Sugarcane Phytoplasma Diseases
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® Functional Plant Science and Biotechnology ©2012 Global Science Books Recent Biotechnological Approaches in Diagnosis and Management of Sugarcane Phytoplasma Diseases Govind P. Rao1* • Smriti Mall2 • Carmine Marcone3 1 Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110012, India 2 Sugarcane Research Station, Kunraghat, Gorakhpur 273008, U.P., India 3 Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, I-84084 Fisciano (Salerno), Italy Corresponding author : * [email protected]; [email protected] ABSTRACT Phytoplasmas have been reported to be associated with two major sugarcane diseases viz., grassy shoot disease (GSD) and white leaf disease (WLD). Sugarcane yellow leaf syndrome is a new syndrome of sugarcane caused by a luteovirus and a phytoplasma. Sugarcane green grassy shoot (SCGGS) and ramu stunt are also associated with sugarcane from Thailand and Papua New Guinea, respectively. GSD and WLD are causing significant economic losses to sugarcane yield and sugar recovery in Asian countries. Both these phytoplasmas have been spreading very rapidly to newer locations with the help of infected seed material and leafhopper vectors. Hence it would be important to diagnose and manage these phytoplasmas at an early stage of sugarcane growth to avoid further spread and significant losses caused by them. Because of unreliable and unspecific symptoms, the identification and characterization of the associated phytoplasma at an early stage of plant growth is problematic and unreliable. The introduction of molecular genetic methods into plant mycoplasmology about 15 years ago greatly improved the diagnosis of phytoplasma infections in plant and insect hosts. PCR offers several advantages over other methods including versatility, relative simplicity, specificity and high sensitivity, which can be increased by a two-step PCR (nested PCR). It has also become possible to differentiate, characterize and classify the phytoplasmas on a phylogenetic basis, using mainly sequence analysis of ribosomal DNA (rDNA). The biotechnological and molecular assays developed in recent years have been utilized at a large scale to characterize these pathogens at an early stage of growth and for screening planting seed materials for possible elimination and management of theses phytoplasma at an early stage of their growth and propagation. We summarize research being conducted on characterization and management of these phytoplasma by novel biotechnological and molecular approaches. _____________________________________________________________________________________________________________ Keywords: characterization, detection, PCR assays, grassy shoot disease, white leaf disease Abbreviations: PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; SCGS, sugarcane grassy shoot; SCWL, white leaf disease; YLS, yellow leaf syndrome CONTENTS INTRODUCTION........................................................................................................................................................................................ 19 PHYTOPLASMAS INFECTING SUGARCANE ....................................................................................................................................... 20 Grassy shoot ............................................................................................................................................................................................ 20 White leaf ................................................................................................................................................................................................ 21 Leaf yellows ............................................................................................................................................................................................ 24 Green grassy shoot disease ...................................................................................................................................................................... 25 Ramu stunt............................................................................................................................................................................................... 25 ALTERNATIVE HOSTS OF SUGARCANE PHYTOPLASMAS.............................................................................................................. 26 MANAGEMENT APPROACH ................................................................................................................................................................... 26 CONCLUSION............................................................................................................................................................................................ 27 REFERENCES............................................................................................................................................................................................. 27 _____________________________________________________________________________________________________________ INTRODUCTION survive in their eggs. Phylogenetic studies have shown that the common ancestor of phytoplasmas is Acholeplasma Phytoplasmas are phytopathogenic prokaryotes that were laidlawii. Also, phytoplasmas and acholeplasmas use the formerly named as mycoplasma-like organism (MLOs) and conventional UGG codon for tryptophan (Trp) and retain were discovered in Japan in 1967 (Doi et al. 1967). They UGA as a stop codon, while most other mollicutes use were observed in ultrathin sections of plant phloem tissue UGA as a Trp codon. In infected plants, phytoplasmas live and named MLOs because they physically resembled myco- mainly or perhaps only, in the sieve cells of phloem tissue. plasmas. Later, these microorganisms were renamed phyto- The sieve cells are highly specialized cells that are living plasmas in 1994 at the 10th Congress of the International but do not contain nuclei when they are mature. Infection of Organization of Mycoplasmology (1993). Phytoplasmas are a plant by a phytoplasma often results in disease. Symptoms wall-less pleomorphic bacteria with sizes ranging from 200 that are characteristic of phytoplasma diseases include yel- to 800 nm, which could survive and multiply only in plant lowing and reduced size of leaves, stunting of the plant, and phloem or insect haemolymph. They are strictly host- proliferation of axillary buds. Such growth from axillary dependent, but could multiply in insect vectors and also buds often results in a witches' broom appearance. Other Received: 25 June, 2011. Accepted: 22 February, 2012. Invited Review Functional Plant Science and Biotechnology 6 (Special Issue 2), 19-29 ©2012 Global Science Books symptoms may be dieback (death) of terminal portions of than those that used ELISA, and RFLP analysis allowed the branches on trees and other woody plants, poor growth of accurate identification of different strains and species of roots, and bunchy growth at ends of branches. Being molli- phytoplasma. More recently, techniques have been deve- cutes, phytoplasmas lack cell walls and instead are bound loped that allow assessment of the level of infection. Both by a triple layered membrane. The cell membranes of all qPCR and bioimaging have been shown to be effective phytoplasmas studied so far usually contain a single im- methods of quantifying the titre of phytoplasmas within the munodominant protein (of unknown function) that makes plant (Christensen et al. 2004). up the majority of the protein content of the cell membrane A consensus for naming novel phytoplasmas was re- (Christensen et al. 2004). commended by the IRPCM Phytoplasma/Spiroplasma Wor- The typical phytoplasma exhibits a pleiomorphic or king Team-Phytoplasma Taxonomy Group that a ‘Candi- filamentous shape and is < 1 μm in diameter. Like other datus Phytoplasma’ species description should refer to a prokaryotes, DNA is free in the cytoplasm. Phytoplasmas single, unique 16S rRNA gene sequence that has <97.5% cannot be cultured in vitro and thus create a major similarity to that of any previously described ‘Ca. Phyto- drawback in their diagnosis and characterization. Due to the plasma’ species. However, because of the highly conserved lack of molecular characterization of phytoplasmas in ear- nature of the 16S rDNA and of the not uncommon presence lier days, their taxonomy was mainly based on symptoms, of 16S rDNA interoperon sequence heterogeneity, the clas- host-parasite interactions, host range and insect vector. sification based on ‘Candidatus’ or on 16S ribosomal group Microscopic methods including transmission electron does not always provide the molecular distinction necessary microscopy (TEM) and light microscopy have been used to for phytoplasma strain characterization. Moreover, some detect phytoplasmas, but most sensitive is the DAPI (DNA- additional tools for phylogenetic analyses and finer strain specific-6-diaminido-2-phenylindole) fluorescence micros- differentiation of phytoplasmas such as rp, secY, tuf, groEL copy technique (Deeley et al. 1979). However, these tech- genes, and the 16S-23S rRNA intergenic spacer region niques require tissue fixation. Recently bio-imaging methods sequences have been used as supplementary tools selecting requiring sensitive, specific and non-toxic fluorescent dyes those providing the most useful and reliable taxonomic and the use of confocal or multiphoton