Species Identification Of Meloidogyne Spp. On Kiwi Friut In Zhou Zhi, China

The survey conducted to assess the incidence of root-knot disease on kiwi speciesActinidia deliciosa and Actinidia chinensis in8localities in and around zhouzhi countryThe orchard of kiwi in all the8localities were infected with root-knot nematodes.Therefore, overall incidence of the disease was100%. Locality wise variations in theincidence of the disease were, however, found. Highest frequency (90%) was found inHong Feng village,, Zhou Zhi county, Shaanxi province, closely followed by Si Zhu townlocalities. In both the localities the frequency was80%. In Louguan town Zhou Zhi county,Shaanxi and Sha Long Hu village, Si Zhu town, Zhou Zhi county, Shaanxi provincefrequency of the disease was60%and70%respectively. The frequency in Idem, ZhouZhi county, Shaanxi province was50%. The lowest frequency (40%) was found in Idem,Zhou Zhi county, Shaanxi province.The intensity of the disease on kiwi in these localitiesbased on average gall and egg mass indicate was high in general.Areawise variations were, however, noticed. Both gall index and egg mass index(average) ranged between2-5through3and4. The greatest egg mass and gall indicateswas found in Hong Feng area, the area in which the incidence was also greatest. The galland egg masses indices were4in Idem, Si Zhu and Si zhu longhu areas.. The lowestincides were noticed for Idem area. Thus,the intensity of the disease on kiwi was highestin Hong Feng village, Si Zhu town.A survey was conducted in different localities in and around zhozhi country to assessthe incidence of root-knot disease on kiwi orchard. During the survey of kiwi orchard ineach locality samples of the roots of the plants were collected randomly. Root sampleskept in polythene bags and properly labeled were brought to the laboratory and thoroughlyexamined for the presence of galls.. Gall index (GI) and egg mass index (EMI) weredetermined on the following scale:0=0,1=1-2,2=3-10,3=11-30,4=31-100and5=greaterthan100galls or egg masses per root system (Taylor and Sasser,1978). The frequency ofoccurrence (percentage) of the disease in each locality was calculated by the followingformula: Frequency of occurrence=×100The frequency of occurrence (percentage) of thedisease in each locality was calculated by the following formula: Frequency of occurrence=Number of fields with root-knot nematode infection/Number of fields survey×100. Morphological Characterization Soil and root material from the infected kiwi speciestaken in open field in zhou zhi,shaanxi for species identification. Egg masses were kept inpetri dishes at room temperature in a small amount of water. J2were extracted from soilby sieving and Baermann funnel methods. J2and males were fixed in3%formaldehydeand processed to glycerin by the formalin-glycerin method (Hooper,1970; Golden,1990). Procedures used in measuring and preparing specimens were essentially those ofGolden and Birchfield (1972), except some fixed females were cut and mounted in clearlactophenol solution. Measurements of all stages were made with an ocular micrometerwith measurements in micrometers, unless otherwise stated.Second stage juveniles of M.incognita and M. hapla M. javanica M. arenaria were extracted by Baermann funnelmethods. placed in a Petry dish and poured over with0.9%NaCl to prevent femalebursting. Under dissecting microscope nematodes were isolated using scalpel andnematological needle. Morphological variation made this population particularly difficultto identify, and it was initially thought to represent a new species closely related to M.arenaria, M. incognita and M. javanica, M. hapla. While this population consisted ofamosaic of morphological similarities to several species.Several methods of genomic DNA extraction from different developmental stages ofpopulations of root-knot nematodes species. Meloidogyne javanica, M. incognita,M haplaand M. arenaria, were evaluated. Two assays of DNA fingerprinting viz., RandomAmplified Polymorphic DNA (RAPD) based Polymerase Chain Reaction (PCR) wereused. Among the tested DNA extraction methods, a minipreparation method was the mostefficient, cost and time effective for PCR. Methods used for DNA extraction from singlejuveniles or females were more suitable for RAPD-PCR. Typical DNA products of670,420, or1200bp in size were specifically amplified by PCR when DNA extracts of M.javanica, M. arenaria, or M. incognita, respectively, were used.Accordingly, Meloidogyne species in China could be most reliably identified byusing PCR assay. Using RAPD-PCR primer PA-01had produced RAPD DNA patternswith clear bands that clearly distinguished one species from the others and so allowed theidentification of the three Meloidogyne species. Molecular biology techniques for theidentification of Meloidogyne spp. could be particularly useful in cases of mixedpopulations of the three species and as a reliable quarantine tests. To develop a rapid andsensitive method for the detection and identification of Meloidogyne incognita, M.javanica and M. arenaria, four and three random amplified polymorphic DNA(RAPD)fragments specific for M. incognita and M. javanica, respectively, were identified.Based on the sequences of these RAPD markers, were designed and tested for their amplification specificity and efficiency against populations of M.incognita, M.j avanica,M. ar enaria, M. hapla. This resulted in thr ee pairs of PCR primers that w ere used incombination to reliably and sensitively identify M. incognita, M. j avanica and M.arenaria. The PCR markers can be readily amplified from one third of a single second-stage juvenile, maleor female, thus demonstrating that the RAPD based PCR assays havepractical value for routine identification of M. incognita, M. j avanica and M. arenariaM.hapla in soil and root samples.