Mechanisms Of Resistance To Meloidogyne Incognita In Sour Cucumber (Cucumis Hystrix Chakr.) And Identification Of Introgression Lines With Resistance

The root-knot nematode Meloidogyne incognita is an important pathogen and causes significant yield losses to cucumber production throughout the world. Breeding for resistance to M.incognita in cucumber was lagged behind other crops resulting from the poverty of resistance in cultivated cucumber. Introducing resistance in wild into cultivated cucumber based on hybridization is an effective way to make breakthrough in M.incognita-nematode breeding of cucumber. A successful cross has been made between Sour cucumber and cultivated cucumber and Sour cucumber is the important genetic resources for cucumber improvement through interspecific hybridization. In order to exploiting cucumber gene resources resistant to M.incognita, Sour cucumber, cultivated cucumber and interspecific progenies derived from the cross between these two species were used as primary research materials in this paper. Resistant to M.incognita and resistance transfer to its progenies in Sour cucumber were studied, and the internal mechanisms of resistance in Sour cucumber were also investigated. Breeding of cucumber-Sour cucumber introgression lines resistant to M. incognita based on interspecific hybridization were conducted and its values in resistance gene mapping and cucumber improvement were analyzed. The main contents and results are as follows:1. Pathogen identification and screening of germplasm for resistance to the root-knot nematode in cucumberThe morphological observation and isozyme phenotypes analysis of the female were conducted for three Meloidogyne spp. samples collected from infected cucumber roots. The results showed that perineal pattern of the female was similar to Meloidogyne incognita and M.javanica. Malate dehydrogenase (MDH) and esterase (EST) phenotypes of the female were the same as M.incognita, thus the collection of Meloidogyne spp. was identified as M. incognita. Screening for suitable host of M. incognita showed that its reproduction on powder fruit tomato varieties was better than red fruit tomato, in summer and autumn better than in winter and spring. The appropriate culture and conservation time of M. incognita was respectively from 45 days to 55 days and within 4 days in vitro at 4℃after inoculation.Fourteen Cucumis germplasms were selected to evaluate their resistance to M.incognita. Cluster and variance analysis showed that they could be clustered 4 groups, i.e. high susceptibility, moderate susceptibility, susceptibility and high resistance. Among of these Cucumis species, two wild germplasms were high resistance, interspecific progenies were resistance, and other species studied were all susceptible to M.incognita. The high resistance to M.incognita in Sour cucumber was transmitted to its progenies through interspecific hybridization.2. Resistant mechanism of sour cucumber for its resistance to the root-knot nematode Meloidogyne incognitaThe differences in invasion and development of the nematode, the anatomy of root and cytology of feeding site were compared by using resistant and susceptible cucumber varieties. The results showed that the percentage of nematode invasion in Sour cucumber was significantly lower than that in Beijingjietou (P<0.01). The individual ratio of female and male nematodes was respectively 1:12 and 1:5 in resistant and susceptible roots (P<0.01). The resistant reaction observed in Sour cucumber could inhibit nematode from feeding and juvenile development. The hypersensitive necrosis of cells occured in resistant reaction, no necrosis could be detected in susceptible one. Limited invasion, inhibited feeding of nematodes and necrosis response of feeding site cells were the main features occured in Sour cucumber displaying resistance to M.incognita.High performance liquid chromatography was used to study the relationships between resistance and content of Cucurbitacin B in cucumber. The results showed that the highest content of Cucurbitacin B in roots before inoculation is in Sour cucumber (1.31μg/g), followed by the Africa Horned cucumber (0.85μg/g) and the minimum Beijingjietou (0.35μg/g), indicating that the ontology content of Cucurbitacin B in Sour cucumber were higher. The content of Cucurbitacin B in these three Cucumis species were increased by 198%,214% and 40% respectively than the corresponding controls after inoculation, indicating the increased content of Cucurbitacin B in Sour cucumber is greater. The infected degree of Sour cucumber is lower indicating its resistance is higher. The correlation analysis showed that there was positively correlation between nematode-resistant abilities of crops and the content of Cucurbitacin B in roots, the higher its content, the stronger the resistance. The resistance in Sour cucumber for its chemical aspects results from its higher levels of Cucurbitacin B in roots.Cucumis genotypes with different resistance to M.incognita were used to study the effects of nematode infection on reactive oxygen metabolism, defense enzyme activities and resistance index related in roots and leaves of cucumber. The results showed that the increase rate of the content of superoxide anion radical (O2·-) and hydrogen peroxide (H2O2) were significantly lower in the resistant genotype Sour cucumber than in the susceptible genotype Beijingjietou. The correlation analysis showed that there was respectively a significant positive correlation of superoxide dismutase (SOD) (r=0.8278*), highly significant negative correlation of peroxidase (POD) (r=-0.9679**) and significant negative correlation of phenylalanine ammonialyase (PAL) (r=-0.8499*) between the rates of changes of SOD, POD and PAL activities in roots associated with the disease index for Cucumis species. POD isoenzyme electrophoresis analysis showed that there were three more enzyme bands (Rf=0.126,0.188,0.944) in Sour cucumber, but only one weak band (Rf=0.396) in Beijingjietou when compared to corresponding control. The rate of increase of EL and MDA induced by M.incognita was higher in Beijingjietou than in Sour cucumber, the root activities were significantly lower in the latter than the former. Oxidative stress infected by M.incognita was less and the root activities were higher in Sour cucumber so that it displayed resistance to M.incognita after infection.The effects of nematode infection on photosynthesis, chlorophyll fluorescence parameters and physiological indices related of cucumber were investigated. The results showed that it induced a significantly lower decrease of chlorophyll (Ch1) content in Sour cucumber leaves than in Beijingjietou. Decreased extents of its content accompany with nitrogen content reduction in Sour cucumber were less than in Beijingjietou. Net photosynthetic rate(Pn), stomatal conductance (Gs) decreased in two varieties, intercellular CO2 concentration (Ci) decreased in Sour cucumber, while its value have a sharp rise in Beijingjietou after fourteen days inoculation. Gs reduction in response to relative water content (RWC) declining in Sour cucumber was more sensitive than in Beijingjietou. The nematode infection led to a lower reduction of actual photochemical efficiency (ΦPSⅡ) and photochemical quenching (qP), while induced a significantly higher increase of non-photochemical quenching (qN) in Sour cucumber than in Beijingjietou. Nematode infection has little effect on the initial slope of Pn/Ci in Sour cucumber, leaving a sharp decrease in Beijingjietou. The results indicated that there are a relatively higher nutrients and water use efficiency, lower decrease of Chl, more light energy for photochemical reactions, stronger the heat dissipation ability, so as to maintain a higher Pn in Sour cucumber compared with nematode-susceptible ones. All these did not cause significant effect on the plant growth of resistant Sour cucumber infected by nematode.3. Breeding of cucumber-sour cucumber introgression lines with resistance to the root-knot nematode Meloidogyne incognitaInterspecific progenies from cross between cucumber and Sour cucumber were screened and an introgression lines ILs-10-1 was obtained for resistance to M. incognita through resistance evaluation. The results showed that introgression lines ILs-10-1 displayed high resistance to M. incognita and its resistance was stable. In agronomic traits and morphology, it was characterized to have small-sized leaf, short fruit, brown spine and multi-branching, and it have affinity of hybridization with the cultivated cucumber. Simple sequence repeat (SSR) analysis was carried out with the genomic DNA of ILs-10-1. A pair of primers SSR18648 screened could repeatedly amplify special DNA fragment from its wild parent Sour cucumber. Thus introgression lines ILs-10-1 was confirmed as an interspecific progenies at molecular level.