Environmental Adaptability Of Soybean Cyst Nematode (Heterodera Glycines)

Soybean cyst nematode (Heterodera glycines Ichinohe, 1952, SCN) is one of the mostdestructive pest of soybean. Because of the special stage-dormancy during its life-history,SCN can overcome stress environment, especially long cold winter. Experiencing longperiods of evolution, SCN is able to adapt to various environments and conditions. Westudied on effects of adverse environmental factors, which were common in soybeancropping, on SCN. In order to reveal the anti-stress mechanism of SCN, many physiologicaland biochemical experiments had been done, and the main results were summarized asfollow:1. The macroelement and microelement compounds were tested against the SCN, in vitroand in vivo on soybean. In vitro, KH₂PO₄ and (NH₄)₂HPO₄ were better treatments forreducing the number of surviving J2 and achieved the lowest value of LC50 of macroelementtreatments. Among microelement treatments, FeCl₃·6H₂O was the best treatment inhibiting J2survival with the lowest value of LC50. In glasshouse, FeCl₃·6H₂O suppressed SCNreproduction obviously and achieved the highest percentage of nematode inhibition. In thefield, all the tested compounds inhibited reproduction of the first generation of SCN, but hadno effect on the second generation. All treatments improved growth of root, and applicationof (NH₄)₂HPO₄ and FeCl₃·6H₂O gave better root growth (dry and fresh weighs) of soybean.2. The effects of eight kinds of herbicides were tested on survival of SCN J2 and eggshatching in the lab. Results showed that acetochlor and 2,4-D+propisochlor were effectiveherbicides on SCN. Mortality of J2 treated with acetochlor reached to the highest of all testedherbicides, and 2,4-D butyl ester+propisochlor was the best treatment to inhibit SCN eggshatching. Experiments in the glasshouse and in the fields showed that acetochlor and2,4-D+propisochlor significantly inhibited reproduction of SCN. These two herbicides had noharmful effect on growth of roots.3. The influence of different pH conditions on mortality of SCN J2 were tested, the resultsshowed that SCN could adapt to wide pH range. We also tested effects of temperature, pHvalue, light and soil water content on dormant SCN. The results showed that pH 9.0 KOH,pH 5.0 H₂SO₄ and pH 6.0 H₂MoO₄ improved eggs hatching of SCN. In addition, SCN J2 hadresistance to low and high temperature, especially to low temperature. Cold and thermalacclimation could improve the cold and thermal tolerant. By analyse the relationship between trehalose concentration in SCN and adverse temperature tolerance, a positive correlationbetween cold tolerance was observed. But we didn't found the obvious correlation betweentrehalose content and thermal tolerance. Eggs hatching rate was improved with increasing oftemperature among 18℃-30℃, higher temperature and long illumination were help to breakdormancy of SCN. In vitro, 15% soil water content was proper to eggs hatching of SCN.4. DNA were extracted from different stages of SCN by means of different methods. Theelectrophoresis results showed that DNA extracted from female adult was better than thatfrom other tested state. As PCR amplification template, DNA extracted from different SCNstate by different tested methods, had no effect on PCR results. DNA sequence of SCN heatshock protein (Hsp70) gene, which length was 2841bp, was obtained from SCN total DNAgenome by genome walking technique. Based on the known 2841bp sequence, SCN Hsp70gene (Hg-Hsp-70) CDS sequence was obtained by RT-PCR, the length was 1950bp and theGenBank accession number was FJ816100. Based on the known SCN heat shock protein(Hsp90) gene sequence, the 3003bp SCN Hsp90 gene (Hg-Hsp-90) sequence was obtained,which GenBank accession number was FJ985783. And the gene CDS was isolated from SCNRNA by RT-PCR. The total length of Hg-Hsp-90 CDS was 2166bp, and the GenBankaccession number was FJ985784.5. Prokaryotic expression vectors of SCN Hsp70 and Hsp90 were construsted bypEASY-E1 and successfully expressed in E.coli. Tansetta (DE3), were namedHg70pEASY-E1 and Hg90pEASY-E1, respectively. And the Hsp70 was also expressed innon-fusion expression system built by vector pET-30. Both expressed Hg-Hsp-70 andHg-Hsp-90 were identified by western blot, the results showed that target proteins wasexpressed successfully.6. With actin gene as the inner control, the expression of Hg-Hsp-70 and Hg-Hsp-90 inSCN suffering from temperature stress and chemical stress were detected bysemi-quantitative RT-PCR. The results showed that over-expression of Hg-Hsp-70 was one ofimportant stress-tolerance mechanism. Expression quantity of both Hg-Hsp-70 andHg-Hsp-90 increased under stresses. The level of these gene-express increasing of SCNstressed by higher temperature was higher than that of SCN stressed by low temperature. Inmost stress treatments, the degree of increaseing of Hg-Hsp-90 expression was lower thanthat of Hg-Hsp-70. By means of microscopic observation, we found the body wall ofchemical-stressed SCN shrinked, and stylets of some stressed SCN breaked through the body obviously. Trehalose content and trehalase activity of stressed and unstressed SCN wereassayed by enzyme-labeled instrument. The results showed that both of trehalose content andtrehalase activity increased after stressed by chemiscals, and trehalose content changedregularily with increasing of treated time, suggesting that SCN adapted to the environmentalstress and resisted to stress with regulating synthesis and decomposition of trehalose.7. The dynamic regulation of metabolites was determined by assaying the physiological andbiochemical substances content of dormant SCN. The results showed that trehalose wasaccumulated during the dormancy period, and total sugar, glycogen and soluble proteinparticipated in metabolism of SCN, providing energy for dormant SCN. Glycerol content wasclosely related to cold tolerance of SCN. Data of amino acid showed that total amino acidscontent increased with the enhance of SCN dormant degree, while the amino acids content ofactive SCN was lower than that of dormant SCN. Contents of free leucine, histidine andisoleucine were related to life activities of dormant SCN, and hydrolysis threonine, serine,glutamate, glycine and alanine were related to metabolism of amino acids. SDS-PAGE resultsshowed that the kinds of total protein did not change between dormant SCN and active SCN.The activity of trehalase, Gpase,PFK,PK,FBP and NAD-SDH during SCN dormant periodwere assayed, the results showed that the activity of trehalase and Gpase activity significantlyincreased when SCN enter into dormant stage. But the activity of PFK,PK,FBP andNAD-SDH in dormant SCN were obviously lower than active SCN.