【作者】 侯晓杰；

【导师】 冉隆贤；

【作者基本信息】 河北农业大学 ， 森林培育， 2010， 博士

【摘要】 本研究从河北省主要枣区为害最为严重的枣缩果病的症状、侵染期及发病期的调查入手,对症状单一的枣缩果病进行了组织内欧文氏杆菌的检测、病原菌的组织分离、病健果实内细菌及真菌种群多样性的PCR-DGGE及分子克隆分析、枣缩果病组织苦味物质的分析及枣缩果病的田间防治等,为河北省枣缩果病病原的确定提供理论基础,筛选有效的化学及生物农药,进一步有效防治枣缩果病的危害和发展。主要研究内容如下：1河北省主要枣区枣缩果病症状、侵染期及发病期研究2007-2009年在河北省阜平县、唐县、赞皇县和行唐县枣区选择试验点进行了枣缩果病的症状、侵染期及发病期的调查试验。河北枣区大面积发生且为害最为严重的枣缩果病的典型症状是：受害果多数先是肩部或少数胴部出现淡黄色斑,然后逐渐扩大,成为淡黄色不规则的凹陷病斑,进而病斑处果肉呈土黄色,松软、萎缩,果柄暗黄色,易提前形成离层,遇雨天、雾天后病果极短时间内大量脱落；未脱落的病果后期病斑处微发黑、皱缩、干瘦,病组织呈海绵状坏死,维管束变褐、味苦、不堪食用,无经济价值。比较阜平、唐县和赞皇枣的发病期发现,调查地点枣缩果病发病时间有差异,阜平的初期病果出现在8月20日左右,8月30日至9月中旬,病果率迅速上升；唐县枣林缩果病发病期较阜平提前5d；赞皇大枣在7月中下旬已发现病果,8月10日病害已呈现中后期症状,比阜平大枣的发病期提前1个月左右。本研究通过套袋试验证明,枣缩果病病菌在果实上的侵入期为7月中旬至8月上旬。2枣缩果病果实内欧文氏杆菌的分子检测通过对河南灰枣、阜平大枣、清苑大枣及唐县婆枣缩果病枣果病部及健部DNA的提取,利用欧文氏菌的特异性引物SR3F和SR1CR,采用聚合酶链式反应(PCR)技术检测欧文氏菌是否存在于枣果实内。结果表明,枣病、建果组织内都扩增出了特异性的欧文氏杆菌的条带。3枣缩果病病原的分离本文于2006年-2009年对采自河北省阜平马房沟村枣区的婆枣、唐县羊角乡枣区的婆枣、赞皇北豪村的赞皇大枣、行唐鳌鱼村的婆枣及河南省孟庄乡灰枣产区的枣缩果病果实进行了病原菌的分离试验,并对其进行了形态学的鉴定和致病性测定。结果表明,缩果病枣果内微生物的分离比率较低,只达14.0%,且分离的菌株大多来自表皮；经培养特征和形态学鉴定,分离比率较高的是交链孢菌真菌(Alternaria),茎点霉菌(Phoma)和小穴壳属(Dothiorella)真菌次之,还有少量的青霉菌、炭疽菌等和其它未鉴定的真菌和3种细菌,且同属的真菌分离物形态差较大。分别选择分离比率较高的交链孢菌、茎点霉菌和小穴壳属的菌株进行柯赫式证病法回接实验,3种真菌都能使枣果实发病,发病症状与林间缩果病症状相似,但通常出现软腐病斑。对林间接种后表现症状的果实进行再分离,均可分离出3种真菌。4枣缩果病果实内微生物种群多样性的PCR-DGGE分析运用聚合酶链式反应(PCR)技术,以阜平大枣枣缩果病果实病部、阜平大枣健康果实抽提到的组织总DNA为模板,对其细菌16S rDNA的V3可变区及真菌18S rDNA进行扩增,并对扩增产物进行DGGE指纹图谱分析,以研究阜平大枣缩果病果实与健康果实内的微生物种群变化。试验结果表明,PCR-DGGE法是研究枣果内微生物组成的可行方法；阜平大枣缩果病果实内细菌及真菌种群较健康果实种类减少,有些细菌优势菌在病部样品内不存在,健部部分优势菌在病部为非优势菌,真菌优势菌群病果较健康果实种类减少。5枣缩果病果实内微生物种群多样性的分子克隆研究本文以枣缩果病发病期采集的病果和健康果实为材料,利用基于PCR技术的分子生物学方法——16S rRNA基因克隆文库的构建(16S rRNA Gene Clone Library)技术,分析阜平大枣缩果病果实、健康果实和河南灰枣缩果病果实、健康果实内的细菌16S rDNA和真菌18S rDNA基因片段多态性,结合克隆、测序,研究了枣病果和健康果实内的细菌和真菌群落结构的变化。对每个样品分别随机挑取的10个经检测后的阳性克隆子进行测序,结果表明：真菌和细菌分别只在河南灰枣病样中得到了两种不同的微生物基因片段。6枣缩果病组织苦味物质的色谱分析本文利用不同极性溶剂水、乙醇、正己烷-乙酸乙酯-丁醇对不同地区采集的枣缩果病果实病斑处苦味物质进行抽提并对其进行高压液相色谱和气-质联用色谱的分析。结果表明,溶剂水、乙酸乙酯和丁醇不适于苦味物质的提取,乙醇和正己烷可以从病果组织内提取出带有明显苦味的物质；5个地区的病样提取物经高压液相色谱分析后,都未发现明显的可以区别于对照样品的峰谱；唐县婆枣、赞皇大枣、阜平大枣、河南灰枣和行唐婆枣样品正己烷提取物经气-质联用色谱仪分析,病样内都出现了不同于健康样品的物质,这些病样内的特殊物质经质谱分析后其化学结构式与所用气-质联用色谱仪内NIST05.L质谱图库中相似性高于90%以上的物质有Hexadecanenitrile(十六烷基腈)、Oleanitrile(油酸腈)、Hexadecanamide(十六酰胺)、9-Octadecenamide(9-十八烯酰胺),其相似性分别为91%、95%、96%、95%,其中Oleanitrile和Hexadecanamide的化学结构式含有苦味基团“≡N”。7枣缩果病防治研究据查阅枣缩果病防治资料和作者前期对枣缩果病病原菌的分离结果,为进一步有效防治枣缩果病的危害和发展,选用药剂和粘着剂共12种物质,组合成9种药剂配方进行了田间药剂试验。结果表明：0.5%NaHCO3+0.25%植物油+洗洁精,高脂膜200倍液+链霉素140单位/毫升+40%氧化乐果1000倍液对枣缩果病的防效最好,其次为高脂膜200倍液+特普唑12.5%可湿性粉剂3000倍液+高效氯氰菊酯3000倍液和六龟裂链霉菌培养液+成膜剂(桃胶：羧甲基纤维素钠=3：1)1000倍液,高脂膜200倍液和多菌灵800倍液有一定的防治效果,但防效不稳定。其它药剂无防效。更多还原

【Abstract】 In this study, the symptom, initial infection period and onset of the jujube fruit shrink disease, the most serious diseases of jujube fruit, were investigated in Hebei Province. Molecular detection of Erwinia carotovora in the tissue of jujube fruit shrink disease, isolation of pathogen, diversity analysis of microbial community from the fruit of jujube fruit shrink disease by PCR-DGGE and molecular cloning, the analysis of bitterness compounds of fruit shrink disease by high pressure liquid chromatography (HPLC) and gas chromatography-mass chromatography(GC/MC), and the control of jujube fruit shrink disease were studied as well. The main results of this research were showed as follows:1 Survey of the symptom, infection period and onset of the jujube fruit shrink disease in the main jujube fields of Hebei ProvinceDuring 2007-2008, the symptom, infection period and development of symptoms of the jujube fruit shrink disease were observed at Fuping, Tang, Zanhuang and Xingtang counties in Hebei Province. The typical symptom of the jujube fruit shrink disease is that firstly the yellow spots appeared the near the parts of jujube fruit pedicel or the middle of the jujube fruit; subsequently, the spots expanded into the irregular sunken and light yellow spots. Over time, sarcocarp of jujube has become yellowish brown, soft and shrink, and the jujube fruit pedicel became dark yellow. A large number of jujube fruits will fall off after rain or heavy fog. The spots became light black, wrinkled and skinny in the late, and the diseased tissues were sponge-like necrosis, vascular browning. Jujube fruit losed the economic value because of the bitterness. In the course of the investigation, we foundind that the beginning of symptom period of jujube fruit shrink disease is almost the same but slightly different in different areas of Hebei Province. There are very few diseased fruits with early symptoms at mafanggou in Fuping county on 20th Aug 2007, in the next few days the development of diseased fruit was slow. On 30th Aug 2007 after two rainy days, we observed the rot development rate increased rapidly, and 80% of the diseased fruit with typical symptoms fell to the groud on 1th Sep 2007, occupying almost 50% of the total amount of fruit. In 2007, compared with the beginning to show symptoms period in Fuping county, jujube fruit shrink disease occurred 5 days earlier in Tang county. Parts of the diseased fruits were found on 20th August, and the diseased rate of fruit showed a gradually increasing trend. In 2008, the diseased fruit were found in mid and late July, and on 10th August, we collected the diseased fruit with late symptom. This study proved that the invasion period of fruit shrink disease pathogen is the mid-July to mid-August. 2 Molecular detection of Erwinia carotovora in the tissue of fruit shrink of jujubeThe DNA of diseased and healthy Z. jujuba var. henanhuizao, Z. jujuba var. fupingdazao, Z. jujuba var. qingyuanhongzao and Z. jujuba var. tangxiandazao, was extracted. Erwinia carotovora was detected by PCR with specific primers SR3F and SR1cR. The results show that, E. carotovora broadly exist in the diseased and healthy fruit of Chinese jujube, indicating that E. carotovora may not be the pathogen of jujube fruit shrink disease.3 Isolation and identification of pathogeny of jujube fruit shrink diseaseIsolation and identification of the pathogeny of jujube fruit shrink disease were carried in Z. jujuba var. fupingdazao, Z jujuba var.tangxianpozao, Z. jujuba var. zanhuangdazao, Z. jujuba var. xingtangpozao and Z. jujuba var. henanhuizao in 2006 and 2009. The results showed that the isolation rate of the fungal microbes was only up to 14.0%, and most of its recovering parts were the epiderm of jujube. Alternaria spp. separation rate is the highest, followed by Phoma spp. and Dothiorella gregaria Sacc.. The lowest isolation rates were Penicillium, Colletotrichum and several unidentified fungal and bacterial strains. Each selected species of Alternaria spp., Phoma spp. and Dothiorella gregaria Sacc, separately, was inoculated on jujube fruit in the field. The symptoms of jujube fruit inoculated with Alternaria spp., Dothiorella gregaria Sacc. and Phoma spp. by stabing were soft rot, and the symptoms were soft rot. The reisolation rares of the three inoculated fungi were 26.7%、13.3% and 6.7%, respectively.4 Diversity analysis of microbial community from the fruit of jujube fruit shrink disease by PCR-DGGEThe V3 variable fragment for 16S rDNA of bacteria and the variable fragment for 18S rDNA of fungi were amplified from the genome DNA by PCR. The DNA extracted from the healthy fruit of Z. jujuba var. fupingdazao and the fruit of jujube fruit shrink disease. The diversity of bacterial communities of Z. jujuba var. fupingdazao was studied by PCR-DGGE. The results showed that the PCR-DGGE technology was a feasible method for diversity analysis of Z. jujuba var. fupingdazao. The population of endophytic bacteria and endophytic fungi from the diseased friuts was fewer than the ones from the healthy friut, and there was little change in their predominant bacterial floras. However, the predominant fungal floras in diseased friuts were fewer than the ones in healthy friuts.5 The study on bacterial and fungal species diversity in fruit of jujube fruit shrink disease by the molecular cloningThe bacterial and fungal community structures of the healthy and diseased fruits of Z. jujuba var. fupingdazao and Z. jujuba var. henanhuizao were mainly studied during the ocurrence of jujube fruit shrink disease using molecular biology methods-16S rRNA gene clone library construction technology. The bacterial 16S rDNA and fungal 18S rDNA gene fragment length polymorphism were analysed in both fruit types with cloning and sequencing method. After sequence 10 tested positive clones randomly picked from eath sample, the results showed that two fungal and bacterial genes sequence existed in diseased fruit of Z. jujuba var. henanhuizao, which indicated that there were few micobial community in jujube fruit.6 The analysis of bitterness of fruit shrink disease by high pressure liquid chromatography (HPLC) and gas chromatography-mass chromatography (GC/MC)The results showed that water, ethyl acetate and butanol were unsuitable for extracting of bitter substances, and ethanol and hexane could extract the material with obvious bitterness from the diseased tissue. The bitterness compounds were analysed by high pressure liquid chromatography, and the peaks were not obviously different between diseased and healthy jujube samples.The hexane extract of Z. jujuba var. tangxianpozao, Z. jujuba var. zanhuangdazao, Z. jujuba var. fupingdazao, Z. jujuba var. henanhuizao and Z. jujuba var. xingtangpozao was analysed by GC/MS, finding different materials in the diseased samples compared with the healthy ones. The extract was compared with hexade-canenitrile, oleanitrile, hexadecanamide and 9-octadecenamide in NIST05.L Mass Gallery, and the result showed that their similarities were reached to 91%,95%,96% and 95%, respectively. And oleanitrile group "=N" with bitter taste exsisted in the chemical structure of hexadecanenitrile and oleanitrile.7 The control of jujube fruit shrink diseaseAccording to data and the isolation results of the jujube fruit shrink diseases pathogen from the initial experiment, nine fungicides were tested in the field. The results showed that the 0.5% NaHCO3+ 0.25% vegetable oils+ family using detergent and the lipid membrane with 200 times dilution+ streptomycin with 140 units/mL+40% omethoate diluted 1000 times have the best control effect, the second was the lipid membranes with 200 times dilution+12.5% Trapp v1 (WP) with 3000 times dilution+cypermethrin with 3000 times dilution and the filtrate of liquid culture medium of Streptomyces rimosus+film forming material with 1000 times dilution (peach gum sodium carboxymethyl cellulose with ration of 3 to 1), film-forming material 200 times dilutoin and carbendazim diluted 800 times had certain control effect.更多还原