枣缩果病病原和防治研究
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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。
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 Province
During 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 jujube
The 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 disease
Isolation 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-DGGE
The 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 cloning
The 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 disease
According 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.
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。
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 Province
During 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 jujube
The 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 disease
Isolation 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-DGGE
The 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 cloning
The 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 disease
According 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.
引文
[1]曲泽洲,王永蕙主编.中国果树志·枣卷[M].北京：中国林业出版社,1993.
[2]刘孟军.枣属植物分类学研究进展[J].园艺学报,1 999,26(5)：302-308.
[3]贺士元,邢其华,尹祖棠,等.北京植物志：上、下册.1992年修订版[M].北京：北京出版社,1993.
[4]徐启聪.中国不同地区地不同枣树品种上枣疯病植原体的分子鉴定及变异分析[D].北京：中国林业科学研究院,2009,1.
[5]杨雷.酸枣种质资源果实营养成份分析及种质评价[D].河北保定：河北农业大学,2004,1.
[6]沈征言.第23届国际园艺学会学术活动简介[J].园艺学报,1991,18(2)：189-192.
[7]肖邦森.毛叶枣优质高效栽培技术[M].中国农业出版社,1999.
[8]郑少泉,黄爱萍,蒋瑞华.台湾印度枣的品种与栽培[J].福建农业科技,1999,3：20-22.
[9]王红梅.台湾青枣组培快繁技术研究[D].北京：北京林业大学,2004,2.
[10]刘孟军主编.枣优质生产技术手册[M].北京：中国农业出版社,2004：24.
[11]韩金声.北方果树病害及其防治[M].天津：天津农业科技出版社,1979.
[12]陈贻金,陈莫林,朱林元.枣缩果病及其防治技术研究[J].林业科技通讯,1989,8：1-5.
[13]郑晓莲,赵光耀,茆正川,等.枣缩果病的诊断初报叨.植物保护,1995,121(2)：19-21.
[14]康绍兰,邸垫平,李兴红,等.枣铁皮病病原鉴定[J].植物病理学报,1998,28(2)：165-171.
[15]徐祥彬,赖童飞,景云飞.山西壶瓶枣缩果病病原菌分离和鉴定[J].植物病理学报,2009,39(3)：225-230.
[16]Jula F. Morton. Fruits of warm climates[M]:Indian Jujube,1987,272-275.
[17]况红玲.中国部分枣产区枣主要果实病害的病原及药剂筛选[D].北京：北京林业大学,2007,1-2.
[18]吴玉柱,季延平,刘会香,等.冬枣黑斑病病原菌的鉴定[[J].中国森林病虫,2005,24(2)：1-3.
[19]邸垫平,康绍兰,彭士琪,等.枣铁皮病发病生理机制的研究[[J].华北农学报,1997 12,168-172.
[20]中国农科院果树研究所.中国果树病虫志[M].北京：农业出版社,1960.
[21]刘惠珍著.中国主要树种病原名录[M].中国林科院林业研究所,1982.
[22]朱本明.枣树花叶病病原研究报[J].自然杂志,1982,59(1)：77-78.
[23]束庆龙,刘世骇.安徽枣树病害调查研究初报[J].安徽农学院学报,1990,1：37-41.
[24]刘俊展,张路生,常慧红,等.冬枣嫩梢焦枯病病原初步鉴定[[J].植物病理学报,2005,(6)：129-132.
[25]姜娜.枣(Zizyphus jujuba Mill.)铁皮病有效药剂筛选[J].河北保定：河北农业大学,2007,1,43-44.
[26]邵立平主编.真菌分类学[M].北京：中国林业出版社,1984：340.
[27]陈作义等.枣疯病病原体的电子显微镜研究-Ⅱ[J].类菌质体.科学通报,1978,(12)：10.
[28]郑晓莲,卢守信.枣锈病越冬夏抱子萌发及侵染的研究简报[J].植物保护,1992,(3)：21.
[29]常聚普.枣轮纹病发病规律及综合防治技术[J].中国果树,2004,4：29-30.
[30]曲俭绪,沈瑞祥,李志清,等.枣黑腐病病原研究[J].森林病虫通讯,1992,2：1-4.
[31]孙永安,师宗舜,王煨时.大枣炭疽病的研究[J].中国果树,1984,(2)：40-44,50.
[32]林忠敏,赵晓军,赵可俊,等.红枣果实黑斑病的病原分离和鉴定[J].山西农业科学,2001,29(1)：74-77.
[33]辛玉成,王贵禧,崔卫东,等.沾化冬枣果实病害的发生与生态相关性研究初报[J].莱阳农学院学报,2003,20(4)：255-257.
[34]刘春琴,王庆雷,张立震,等.金丝小枣浆烂果病症状,危害及病菌鉴定[J].中国农业大学学报,2004,9(2)：31-35.
[35]张立震,刘春琴,孙玉英,等.金丝小枣果实病害病原菌研究[J].林业科学,2004,40(6)：190-194.
[36]郑晓莲,赵光耀,齐秋锁,等.枣干腰缩果病症状类型及侵染规律研究[J].植物保护,1998,24(4)：17-19.
[37]杨自民,等.枣缩果病的发生原因及防治方法探讨[J].北方果树,2002,(4)：33.
[38]王军,辛玉成,李宝笃,等.沾化冬枣果实黑斑病及防治初报叨.中国果树,2005(2)：10-13.
[39]李晓军,徐颖,阴忠.50%轮纹宁可湿性粉剂防治鲁北冬枣黑斑病和枣果浆烂病试验[J].落叶果树,2004,1：53-54.
[40]苏安仁,王秀荣.枣浆病及其防治技术[J].落叶果树,1994,2：23.
[41]阎振华.枣轮纹烂果病发生及防治[J].北方果树,1997,2：37-38.
[42]王庆雷,刘春琴,张立震,等.金丝小枣浆烂果病侵染循环研究[J].河北农业大学学报,2004,27(5)：63-67.
[43]况红玲.中国部分枣产区枣主要果实病害的病原及药剂筛选[D].北京：北京林业大学,2007,6：2-4,35-36.
[44]周绪宝.冬枣采后黑腐病病害及防治技术的研究[D].北京.中国农业大学,2003,6：14.
[45]郑晓莲,齐秋锁,赵光耀,等.枣缩果病初侵染来源的初步研究[J].河北农业大学学报,1995,18(4)：59-63.
[46]郑晓莲,齐秋锁,赵光耀,等.枣缩果病病原子实体诱导和鉴定[J].植物保护,1 996,22(6)：6-8.
[47]郑晓莲,赵光耀,武丽芬,等.枣缩果病主要病原生物学特性的初步研究[J].植物保护,1996,22(1)：13-16.
[48]李志清,常聚普,乔趁峰,等.枣黑腐病发病规律研究[J].果树科学,1 997,14(4)：252-256.
[49]康绍兰,邸垫平,李兴红,等.枣铁皮病病原鉴定[J].河北农业大学学报,1997,20(1)：90-91.
[50]陈谟林,马元忠,李占林等.枣缩果病发病过程及流行规律[J].河南科技,1996,4：11-12.
[5l]李京涛,陈素珍,王海臣.枣缩果病防治药效对比试验[J].河北林业科技,2004,4：10.
[52]康绍兰,李兴红,邸垫平,等.枣铁皮病发病规律的研究[J].河北农业大学学报,1997,20(增刊)：5-19.
[53]罗学平.枣缩果病防治试验初报[J].落叶果树,2003,6：51-52.
[54]陈贻金,陈谟林,朱林元.枣缩果病及其防治技术研究[J].林业科技通讯,1989,8：1251.
[55]毛永民.防治“枣铁病”特效药剂研制成功[J].农村科技开发,1997,5)：19.
[56]艾庆芝,付秀芝,王文清.特谱唑防治枣树缩果病试验初报[J].落叶果树,199,3：25.
[57]宋宏伟,宋小菊,刘俊磊,等.12种杀菌剂防治枣缩果病田间药效试验报告[J].河南林业科技,1999,19(3)：9211.
[58]刘惠娟,刘贤谦,刘随存,等.7种药剂对枣缩果病病原菌_聚生小穴壳菌的抑制作用[J].山西农业科学,2007,35(3)：66-68.
[59]赵永强.烟草根黑腐病菌的分子检测与室内药剂筛选[D].山东济南：山东农业大学,2009：12-13.
[60]刘玮琦.保护地土壤细菌和古菌群落多样性分析[D].北京：中国农业科学院,2008,12-13.
[61]Fischer S.G, Lerman L. S., et al. Length independent separation of DNA restriction fragments in two dimensional gel electrophoresis[J]. Cell,1979,16:191-200.
[62]Muyzer G., de Waal E. C., Uitterlinden A. G, et al. Profiling of complex microbial population by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J]. Applied Environmental Microbiology,1993,59(3):23-32.
[63]Xing D.. Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE) [J]. Science in China Ser. C Life Sciences,2005,48(2):155-162.
[64]Garbera P., van Overbeek L. S., van Vuurde J. W. L., et al. Analysis of endophytic bacterial communities of potato by planting and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA basad PCR fragments[J]. Microbial Ecology,2001,41:369-383.
[65]Sessitsch A., Reiter B., Pfeifer U., et al. Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes[J]. FEMS Microbiology Ecology,2002,39:23-32.
[66]Kuklinsky-Sobral J., Araujo W. L., Mendes R., et al. Isolation and characterization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide[J]. Plant and Soil,2005,273:91-99.
[67]Claudia S.G. Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18S rDNA sequence analysis[J]. Journal of Microbiological Methods,2001(47): 345-354.
[68]Eeva J. V., Jarkko H.. Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplied ribosomal DNA[J]. Mycological Research,2000,104 (8):927-936.
[69]Borneman J. and Hartin R. J.. PCR primers that amplify fungal rRNA genes from environmental samples[J]. Applied and Environmental Microbiology,2000,66(10):4356-4360.
[70]Gomes N. C. M., Fagbola O., Costa R., et al. Dynamics of fungal communities in bulk and maize rhizosphere soil in the tropics[J]. Applied and Environmental Microbiology,2003,7:3758-3766.
[71]Wilms R., Sass H., Kopke B., et al. Specific bacterial, archaeal, and eukaryotic communities in tidal-flat sediments along a vertical profile of several meters[J]. Applied and Environmental Microbiology,2006,72(2):2756-2764.
[72]孙磊.非培养方法和.涪养方法对水稻内生细菌和根结合细菌的研究[D].北京：首都师范大学,11,14.
[73]Massol-Deya A. A., Odelsonn D. A., Hickey R. F., et al. Bacterial community fingerprinting of amplified 16S andl6-23S ribosomal DNA gene sequence and restriction endonuclease analysis (ARDRA), In:Akkermans A. D. L., Van Elsas J. D. and De Bruijn F. J.,et al. Molecular Microbial Ecology Manual[M], Kluwer Academic Press, Dordrecht, The Netherlands,1995,1-8 section 3.3.2.
[74]Liu W. T., Marsh T. L., Cheng H., et al. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA[J]. Applied Environmental Microbiology,1997, 63:4516-4522.
[75]Muyzer G, de Waal E. C., Uitterlinden A. G. Profiling of complex microbial population by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J]. Applied Environmental Microbiology,1993,59(3):23-32.
[76]Heuer H., Smalla k.. Evaluation of community-level catabolic profiling using BIOLOG GN microplatcs to study microbial community changes in potato phyllosphere[J]. Journal Microbiology Methods,1997,30:49-61.
[77]Idris R., Trifonova R., Puschenreiter M., et al..Bacterial Communities Associated with Flowering Plants of the Ni Hyperaccumulator Thlaspi goesingense[J]. Applied Environmental Microbiology,2004,70:2667-2677.
[78]Giovannoni S. J., Britschgi T. B., Moyer C. L., et al. Genetic diversity in Sargasso Sea bacterioplankton[J]. Nature, 1990,345:60-63.
[79]Kemp P. F., Aller J. Y. Bacterial diversity in aquatic and other environments:what 16S rDNA libraries and tell us[J]. FEMS Microbiology Ecology,2004,47:167-177.
[80]章元寿.植物病理生理学[M].南京：江苏科技出版社,1995,51-52.
[81]董汉松.植物诱导抗病性的原理和研究[M].北京：科学出版社,1995.
[82]罗孟军,朱天辉.植物病原真菌毒素[J].四川林业科技,2001,22(3)：45-49.
[83]董金皋,李树正.植物病原真菌毒素研究进展[M].北京：中国科学技术出版社,1978.
[84]祁高富,杨斌,叶建仁.植物病原真菌研究进展[J].南京林业大学学报,2003,24(2)：66-70.
[85]Yoder O C. Toxins in pathogenesis Ann. Rev.. Phytopathol.1980,18:103-129.
[86]叶建仁,祁高富.松针褐斑病毒毒素的专化性研究[J].南京林业大学学报,1999,23(6)：1-4.
[87]裘维蕃.菌物学大全[M].科学出版社,1998.
[88]马丽艳,赵耀伟,崔宗士.疫霉菌毒素研究进展叨.东北农业大学学报,2004,35(2)：234-238.
[89]张淑珍.植物病原真菌研究进展[J].黑龙江农业科学,2001,(2)：42-43.
[90]杨斌,叶建仁,包宏.林木病原菌毒素研究进展[J]. Forest Research,2000,3:316-322.
[91]刘胜毅,张建坤.毒素草酸的致病作用与防治策略[A].见：董金皋,李树正.植物病原真菌毒素研究进展(第一卷)[M].北京：中国科技出版社,1997,164-167.
[92]陈捷.植物病原茵毒素的致病机理[A].见：董金皋,李树正.植物病原真菌毒素研究进展(第一卷)[M].北京：中国科技出版社,1997,32-50.
[93]Morooka N., Minowa N., Tsunoda H., et al. Metabolite of a mycotoxin by plant tissue culture-toxicological and chemical studies of diterpene fungal product[C]. Proceedings of the Japanese Association of Mycotoxicology, 1987,26:27-30.
[94]李树正,岳东霞,刘准,等.交链孢酸的提纯与结构鉴定[A].董金皋,李树正.植物病原菌毒素研究进展(第一卷)[C].北京：中国科学技术出版社,1997,218-222.
[95]张利辉,董金皋.植物病原真菌毒素的分离、纯化技术[J].现代科学仪器,2002,2：56-59.
[96]李蕾蕾.苦味食品概述[J].中国食物与营养,2006,6：50-51.
[97]丁耐克.食品风味化学[M].北京：中同轻工业出版社,1996.
[98]朱国斌,鲁红军.食品风味原理与技术[M].北京：北京大学出版社,1996.
[99]曹雁平.食品调味技术[M].北京：化学工业出版社,2002.
[100]夏延斌,甄增立,谢笔钧.食品化学[M].北京：中国轻工业出版社,2001.
[101]韩玉杰,李志西,杜双奎.红枣酶解法提汁工艺研究[J].食品科学,2003,24(4)：85-87.
[102]刘德全.新型保健饮品-------金丝枣汁的生产[J].农业科技通讯,1998,12：27.
[103]刘新华.红枣浸出液的脱苦研究[J].食品科技,2006,9：84-87.
[104]魏象廷.中国枣果实病害研究进展[J].西北农业学报,2006,15(1)：88-94.
[105]徐安传,罗文富,杨艳丽,赵海燕.马铃薯晚疫病种薯带菌的分子检测[J].2004,18(2)：73-76.
[106]刘元荣,陈贻金,等.枣树新病害缩果病防治研究[J].河南农业科学,1988,8：10-11.
[107]李永,田国忠,朴春根,等.我国几种植物植原体的快速分子鉴别与鉴定的研究[J].植物病理学报,2005,35(4)：293-299.
[108]王胜坤,王军,徐大平.四种桉树青枯菌DNA提取方法及PCR检测灵敏度比较[J]. Frest Pest and Disease Sept,2007,.5：4-7.
[109]邵雪玲,等.生物化学与分子生物实验指导.武汉大学出版社.2003：36-37
[110]Toth I. K., Hyman L. J. and Wood J. R.. A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants[J]. The Society for Applied Microbiology,1999,87, 158-166.
[111]王丽花,等.彩色马蹄莲欧文氏菌PCR检测[J].植物保护,2006,32(6)：75-78.
[112]魏景超.真菌鉴定手册[M].上海科学技术出版社,1979.
[113]Groves A. B.. A study of the sooty blotch disease of apples and the causal fungus Gloeodes pomigena. Va. Agric. Exp. Stn. Bull.,1933,50:1-43.
[114]Heuer H., Krsek M., Baker P., et al. Analysis of Actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients[J]. Applied and Environmental Microbiology,1997,63:3233-3241.
[115]Gomes N. C., Fagbola O., Costa R., et al. Dynamics of fungal communities in bulk and maize rhizosphere soil in the tropics[J]. Applied and Environmental Microbiology,2003..69:3758-3766.
[116]赵勇,李武,周志华,等.应用PCR-RFLP及PCR-TGGE技术监测农田土壤微生物短期动态变化[J1.南京农业大学学报,2005,28(3)：53-57.
[117]李延茂,胡江春,张晶,等.杉木连栽土壤微生物多样性的比较研究[J].应用生态学报,2005,16(7)：1276-1278.
[118]宋亚娜,包兴国,李隆等.利用DGGE法研究不同种植体系中根际微生物群落结构[J].生物学杂志,2006,23(5)：12-15,4.
[119]周琳,张晓君,李国勋,等.DGGE/TGGE技术在土壤微生物分子生态学研究中的应用[J].生物技术通报,2006,5：67-71.
[120]唐影.转基因抗虫棉对根区土壤细菌多样性影响的初步研究[D].中国农业科学院,2007.
[121]罗海峰,齐鸿雁,薛凯等.PCR-DGGE技术在农田土壤微生物多样性研究中的应用[J].生态学报,2003,23(8)：1570-1575.
[122]Teske A., Wawer C., Muyzer G. Distribution of sulfate-reducing bacteria in a stratified fjord(Mariager Fjord,Denmark)as evaluated by most-probable-number counts and denaturing gradient gel electro-phoresis of PCR-amplified ribosomal DNA fragments[J]. Applied and Environmental Microbiology,1996,62:1405-1415.
[123]Ferris M. J., Muyzer G, Ward D. M.. Denaturing gradient gel electrophor-esis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community[J]. Applied and Environmental Microbiology,1996, 62(2):340-346.
[1241李沁元,崔晓龙,张东华,等.云南腾冲热海两热泉菌藻席细菌多样性的研究[J].微生物学报,2004,44(4)：431-435.
[125]王涛,柴丽红,崔晓龙,等.免培养法对一热泉细菌多样性的初步研究[J].微生物学报,2003,43(5)：541-546.
[126]Eichner C. A., Erb R. W., Timmis K. N., et al. Thermal gradient gel electrophoresis analysis of bioprotection from pollutant shocks in the activated sludge microbial community[J]. Applied and Environmental Microbiology,1999, 65:102-109.
[127]Tsai Y. L., Olson B. H.. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction[J]. Applied and Environmental Microbiology,1992,58:2292-2295.
[128]Sekiguchi H., Watanabe M., Nakahara T. Succession of bacterial community structure along the Changjiang River determined by denaturing gradient gel electrophoresis and clone library analysis[J]. Applied and Environmental
Microbiology,2002,68(10):5142-5150.
[129]向少能,陈琳,何晓丽,等.水体微生物多样性PCR-DGGE分析方法的比较研究[J].重庆工学院学报(自然科学版),2007,7：74-78,97.
[130]Zoetendal E. G, Akkermans A. D., De Vos W. M.. Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria[J]. Applied and Environmental Microbiology,1998,64(10):3854-3859.
[131]Simpson J. M., Mccracken V. J., White B. A.. Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota[J]. Journal of Microbiological Methods,1999,36(3):167-179.
[132]卓凤萍.贡嘎蝠蛾幼虫肠道细菌多样性分析[D].重庆大学,2005.
[133]庞小燕,张宝让,魏桂芳等.应用TGGE技术分析人肠道中双歧杆菌的组成[J].微生物学报,2005,45(5)：738-743.
[134]Vainio E. J. and Hantula J.. Direct analysis of wood-inhabiting fungi using denaturinggradient gel electrophoresis of amplified ribosomal DNA[J]. Mycological Research,2000,104 (8):927-936.
[135]龙良鲲,羊宋贞,姚青,等.AM真菌DNA的提取与PCR-DGGE分析[J].菌物学报,2005,24(4)：564-569.
[136]沈洪,陈明杰,赵永昌,等.羊肚菌内生细菌DGGE鉴定[J].上海农业学报,2008,24(2)：58-60.
[137]梁新乐,朱扬玲,蒋予箭,等.PCR-DGGE法研究泡菜中微生物群落结构的多样性[J].中国食品学报,2008,8(3)：133-137.
[138]工海燕,张晓君,徐岩,等.浓香型和芝麻香型白酒酒酷中微生物菌群的研究[J].酿酒科技,2008,2：86-89,91.
[139]雷娟利,周艳,丁桔,等.不同蔬菜连作对土壤细菌DNA分子水多态性形响的研究[J].中国农业科学,2005,38(10)：2076-2083.
[140]王蒙.长江口九段沙湿地盐沼植物根围细菌群落结构和多样性的研究[D].上海,复旦大学,2006,52.
[141]Schabereiter-Gurtner C., Pinar G, Lubitz W., et al. Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18S rDNA sequence analysis[J]. J Microbiol Methods,2001,47:345-354.
[142]蔡莹.对虾养殖沉积环境细菌多样性的分子分析与若干可培养技术优化探讨[D].厦门,厦门大学,200
[143]原贵生,刘惠娟,刘贤谦等.七种药剂对枣缩果病病原菌—细交链孢菌抑制作用[J].2007,26(6)：28-30.
[144]刘惠娟,刘随存,刘贤谦,等.枣缩果病与果实生理指标关系的研究[J].山西林业科技,2007,1：19-21,32.
[145]屈建华.枣缩果病的克星—靓枣[J].果农园地,2009,1：48.
[2]刘孟军.枣属植物分类学研究进展[J].园艺学报,1 999,26(5)：302-308.
[3]贺士元,邢其华,尹祖棠,等.北京植物志：上、下册.1992年修订版[M].北京：北京出版社,1993.
[4]徐启聪.中国不同地区地不同枣树品种上枣疯病植原体的分子鉴定及变异分析[D].北京：中国林业科学研究院,2009,1.
[5]杨雷.酸枣种质资源果实营养成份分析及种质评价[D].河北保定：河北农业大学,2004,1.
[6]沈征言.第23届国际园艺学会学术活动简介[J].园艺学报,1991,18(2)：189-192.
[7]肖邦森.毛叶枣优质高效栽培技术[M].中国农业出版社,1999.
[8]郑少泉,黄爱萍,蒋瑞华.台湾印度枣的品种与栽培[J].福建农业科技,1999,3：20-22.
[9]王红梅.台湾青枣组培快繁技术研究[D].北京：北京林业大学,2004,2.
[10]刘孟军主编.枣优质生产技术手册[M].北京：中国农业出版社,2004：24.
[11]韩金声.北方果树病害及其防治[M].天津：天津农业科技出版社,1979.
[12]陈贻金,陈莫林,朱林元.枣缩果病及其防治技术研究[J].林业科技通讯,1989,8：1-5.
[13]郑晓莲,赵光耀,茆正川,等.枣缩果病的诊断初报叨.植物保护,1995,121(2)：19-21.
[14]康绍兰,邸垫平,李兴红,等.枣铁皮病病原鉴定[J].植物病理学报,1998,28(2)：165-171.
[15]徐祥彬,赖童飞,景云飞.山西壶瓶枣缩果病病原菌分离和鉴定[J].植物病理学报,2009,39(3)：225-230.
[16]Jula F. Morton. Fruits of warm climates[M]:Indian Jujube,1987,272-275.
[17]况红玲.中国部分枣产区枣主要果实病害的病原及药剂筛选[D].北京：北京林业大学,2007,1-2.
[18]吴玉柱,季延平,刘会香,等.冬枣黑斑病病原菌的鉴定[[J].中国森林病虫,2005,24(2)：1-3.
[19]邸垫平,康绍兰,彭士琪,等.枣铁皮病发病生理机制的研究[[J].华北农学报,1997 12,168-172.
[20]中国农科院果树研究所.中国果树病虫志[M].北京：农业出版社,1960.
[21]刘惠珍著.中国主要树种病原名录[M].中国林科院林业研究所,1982.
[22]朱本明.枣树花叶病病原研究报[J].自然杂志,1982,59(1)：77-78.
[23]束庆龙,刘世骇.安徽枣树病害调查研究初报[J].安徽农学院学报,1990,1：37-41.
[24]刘俊展,张路生,常慧红,等.冬枣嫩梢焦枯病病原初步鉴定[[J].植物病理学报,2005,(6)：129-132.
[25]姜娜.枣(Zizyphus jujuba Mill.)铁皮病有效药剂筛选[J].河北保定：河北农业大学,2007,1,43-44.
[26]邵立平主编.真菌分类学[M].北京：中国林业出版社,1984：340.
[27]陈作义等.枣疯病病原体的电子显微镜研究-Ⅱ[J].类菌质体.科学通报,1978,(12)：10.
[28]郑晓莲,卢守信.枣锈病越冬夏抱子萌发及侵染的研究简报[J].植物保护,1992,(3)：21.
[29]常聚普.枣轮纹病发病规律及综合防治技术[J].中国果树,2004,4：29-30.
[30]曲俭绪,沈瑞祥,李志清,等.枣黑腐病病原研究[J].森林病虫通讯,1992,2：1-4.
[31]孙永安,师宗舜,王煨时.大枣炭疽病的研究[J].中国果树,1984,(2)：40-44,50.
[32]林忠敏,赵晓军,赵可俊,等.红枣果实黑斑病的病原分离和鉴定[J].山西农业科学,2001,29(1)：74-77.
[33]辛玉成,王贵禧,崔卫东,等.沾化冬枣果实病害的发生与生态相关性研究初报[J].莱阳农学院学报,2003,20(4)：255-257.
[34]刘春琴,王庆雷,张立震,等.金丝小枣浆烂果病症状,危害及病菌鉴定[J].中国农业大学学报,2004,9(2)：31-35.
[35]张立震,刘春琴,孙玉英,等.金丝小枣果实病害病原菌研究[J].林业科学,2004,40(6)：190-194.
[36]郑晓莲,赵光耀,齐秋锁,等.枣干腰缩果病症状类型及侵染规律研究[J].植物保护,1998,24(4)：17-19.
[37]杨自民,等.枣缩果病的发生原因及防治方法探讨[J].北方果树,2002,(4)：33.
[38]王军,辛玉成,李宝笃,等.沾化冬枣果实黑斑病及防治初报叨.中国果树,2005(2)：10-13.
[39]李晓军,徐颖,阴忠.50%轮纹宁可湿性粉剂防治鲁北冬枣黑斑病和枣果浆烂病试验[J].落叶果树,2004,1：53-54.
[40]苏安仁,王秀荣.枣浆病及其防治技术[J].落叶果树,1994,2：23.
[41]阎振华.枣轮纹烂果病发生及防治[J].北方果树,1997,2：37-38.
[42]王庆雷,刘春琴,张立震,等.金丝小枣浆烂果病侵染循环研究[J].河北农业大学学报,2004,27(5)：63-67.
[43]况红玲.中国部分枣产区枣主要果实病害的病原及药剂筛选[D].北京：北京林业大学,2007,6：2-4,35-36.
[44]周绪宝.冬枣采后黑腐病病害及防治技术的研究[D].北京.中国农业大学,2003,6：14.
[45]郑晓莲,齐秋锁,赵光耀,等.枣缩果病初侵染来源的初步研究[J].河北农业大学学报,1995,18(4)：59-63.
[46]郑晓莲,齐秋锁,赵光耀,等.枣缩果病病原子实体诱导和鉴定[J].植物保护,1 996,22(6)：6-8.
[47]郑晓莲,赵光耀,武丽芬,等.枣缩果病主要病原生物学特性的初步研究[J].植物保护,1996,22(1)：13-16.
[48]李志清,常聚普,乔趁峰,等.枣黑腐病发病规律研究[J].果树科学,1 997,14(4)：252-256.
[49]康绍兰,邸垫平,李兴红,等.枣铁皮病病原鉴定[J].河北农业大学学报,1997,20(1)：90-91.
[50]陈谟林,马元忠,李占林等.枣缩果病发病过程及流行规律[J].河南科技,1996,4：11-12.
[5l]李京涛,陈素珍,王海臣.枣缩果病防治药效对比试验[J].河北林业科技,2004,4：10.
[52]康绍兰,李兴红,邸垫平,等.枣铁皮病发病规律的研究[J].河北农业大学学报,1997,20(增刊)：5-19.
[53]罗学平.枣缩果病防治试验初报[J].落叶果树,2003,6：51-52.
[54]陈贻金,陈谟林,朱林元.枣缩果病及其防治技术研究[J].林业科技通讯,1989,8：1251.
[55]毛永民.防治“枣铁病”特效药剂研制成功[J].农村科技开发,1997,5)：19.
[56]艾庆芝,付秀芝,王文清.特谱唑防治枣树缩果病试验初报[J].落叶果树,199,3：25.
[57]宋宏伟,宋小菊,刘俊磊,等.12种杀菌剂防治枣缩果病田间药效试验报告[J].河南林业科技,1999,19(3)：9211.
[58]刘惠娟,刘贤谦,刘随存,等.7种药剂对枣缩果病病原菌_聚生小穴壳菌的抑制作用[J].山西农业科学,2007,35(3)：66-68.
[59]赵永强.烟草根黑腐病菌的分子检测与室内药剂筛选[D].山东济南：山东农业大学,2009：12-13.
[60]刘玮琦.保护地土壤细菌和古菌群落多样性分析[D].北京：中国农业科学院,2008,12-13.
[61]Fischer S.G, Lerman L. S., et al. Length independent separation of DNA restriction fragments in two dimensional gel electrophoresis[J]. Cell,1979,16:191-200.
[62]Muyzer G., de Waal E. C., Uitterlinden A. G, et al. Profiling of complex microbial population by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J]. Applied Environmental Microbiology,1993,59(3):23-32.
[63]Xing D.. Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE) [J]. Science in China Ser. C Life Sciences,2005,48(2):155-162.
[64]Garbera P., van Overbeek L. S., van Vuurde J. W. L., et al. Analysis of endophytic bacterial communities of potato by planting and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA basad PCR fragments[J]. Microbial Ecology,2001,41:369-383.
[65]Sessitsch A., Reiter B., Pfeifer U., et al. Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes[J]. FEMS Microbiology Ecology,2002,39:23-32.
[66]Kuklinsky-Sobral J., Araujo W. L., Mendes R., et al. Isolation and characterization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide[J]. Plant and Soil,2005,273:91-99.
[67]Claudia S.G. Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18S rDNA sequence analysis[J]. Journal of Microbiological Methods,2001(47): 345-354.
[68]Eeva J. V., Jarkko H.. Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplied ribosomal DNA[J]. Mycological Research,2000,104 (8):927-936.
[69]Borneman J. and Hartin R. J.. PCR primers that amplify fungal rRNA genes from environmental samples[J]. Applied and Environmental Microbiology,2000,66(10):4356-4360.
[70]Gomes N. C. M., Fagbola O., Costa R., et al. Dynamics of fungal communities in bulk and maize rhizosphere soil in the tropics[J]. Applied and Environmental Microbiology,2003,7:3758-3766.
[71]Wilms R., Sass H., Kopke B., et al. Specific bacterial, archaeal, and eukaryotic communities in tidal-flat sediments along a vertical profile of several meters[J]. Applied and Environmental Microbiology,2006,72(2):2756-2764.
[72]孙磊.非培养方法和.涪养方法对水稻内生细菌和根结合细菌的研究[D].北京：首都师范大学,11,14.
[73]Massol-Deya A. A., Odelsonn D. A., Hickey R. F., et al. Bacterial community fingerprinting of amplified 16S andl6-23S ribosomal DNA gene sequence and restriction endonuclease analysis (ARDRA), In:Akkermans A. D. L., Van Elsas J. D. and De Bruijn F. J.,et al. Molecular Microbial Ecology Manual[M], Kluwer Academic Press, Dordrecht, The Netherlands,1995,1-8 section 3.3.2.
[74]Liu W. T., Marsh T. L., Cheng H., et al. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA[J]. Applied Environmental Microbiology,1997, 63:4516-4522.
[75]Muyzer G, de Waal E. C., Uitterlinden A. G. Profiling of complex microbial population by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J]. Applied Environmental Microbiology,1993,59(3):23-32.
[76]Heuer H., Smalla k.. Evaluation of community-level catabolic profiling using BIOLOG GN microplatcs to study microbial community changes in potato phyllosphere[J]. Journal Microbiology Methods,1997,30:49-61.
[77]Idris R., Trifonova R., Puschenreiter M., et al..Bacterial Communities Associated with Flowering Plants of the Ni Hyperaccumulator Thlaspi goesingense[J]. Applied Environmental Microbiology,2004,70:2667-2677.
[78]Giovannoni S. J., Britschgi T. B., Moyer C. L., et al. Genetic diversity in Sargasso Sea bacterioplankton[J]. Nature, 1990,345:60-63.
[79]Kemp P. F., Aller J. Y. Bacterial diversity in aquatic and other environments:what 16S rDNA libraries and tell us[J]. FEMS Microbiology Ecology,2004,47:167-177.
[80]章元寿.植物病理生理学[M].南京：江苏科技出版社,1995,51-52.
[81]董汉松.植物诱导抗病性的原理和研究[M].北京：科学出版社,1995.
[82]罗孟军,朱天辉.植物病原真菌毒素[J].四川林业科技,2001,22(3)：45-49.
[83]董金皋,李树正.植物病原真菌毒素研究进展[M].北京：中国科学技术出版社,1978.
[84]祁高富,杨斌,叶建仁.植物病原真菌研究进展[J].南京林业大学学报,2003,24(2)：66-70.
[85]Yoder O C. Toxins in pathogenesis Ann. Rev.. Phytopathol.1980,18:103-129.
[86]叶建仁,祁高富.松针褐斑病毒毒素的专化性研究[J].南京林业大学学报,1999,23(6)：1-4.
[87]裘维蕃.菌物学大全[M].科学出版社,1998.
[88]马丽艳,赵耀伟,崔宗士.疫霉菌毒素研究进展叨.东北农业大学学报,2004,35(2)：234-238.
[89]张淑珍.植物病原真菌研究进展[J].黑龙江农业科学,2001,(2)：42-43.
[90]杨斌,叶建仁,包宏.林木病原菌毒素研究进展[J]. Forest Research,2000,3:316-322.
[91]刘胜毅,张建坤.毒素草酸的致病作用与防治策略[A].见：董金皋,李树正.植物病原真菌毒素研究进展(第一卷)[M].北京：中国科技出版社,1997,164-167.
[92]陈捷.植物病原茵毒素的致病机理[A].见：董金皋,李树正.植物病原真菌毒素研究进展(第一卷)[M].北京：中国科技出版社,1997,32-50.
[93]Morooka N., Minowa N., Tsunoda H., et al. Metabolite of a mycotoxin by plant tissue culture-toxicological and chemical studies of diterpene fungal product[C]. Proceedings of the Japanese Association of Mycotoxicology, 1987,26:27-30.
[94]李树正,岳东霞,刘准,等.交链孢酸的提纯与结构鉴定[A].董金皋,李树正.植物病原菌毒素研究进展(第一卷)[C].北京：中国科学技术出版社,1997,218-222.
[95]张利辉,董金皋.植物病原真菌毒素的分离、纯化技术[J].现代科学仪器,2002,2：56-59.
[96]李蕾蕾.苦味食品概述[J].中国食物与营养,2006,6：50-51.
[97]丁耐克.食品风味化学[M].北京：中同轻工业出版社,1996.
[98]朱国斌,鲁红军.食品风味原理与技术[M].北京：北京大学出版社,1996.
[99]曹雁平.食品调味技术[M].北京：化学工业出版社,2002.
[100]夏延斌,甄增立,谢笔钧.食品化学[M].北京：中国轻工业出版社,2001.
[101]韩玉杰,李志西,杜双奎.红枣酶解法提汁工艺研究[J].食品科学,2003,24(4)：85-87.
[102]刘德全.新型保健饮品-------金丝枣汁的生产[J].农业科技通讯,1998,12：27.
[103]刘新华.红枣浸出液的脱苦研究[J].食品科技,2006,9：84-87.
[104]魏象廷.中国枣果实病害研究进展[J].西北农业学报,2006,15(1)：88-94.
[105]徐安传,罗文富,杨艳丽,赵海燕.马铃薯晚疫病种薯带菌的分子检测[J].2004,18(2)：73-76.
[106]刘元荣,陈贻金,等.枣树新病害缩果病防治研究[J].河南农业科学,1988,8：10-11.
[107]李永,田国忠,朴春根,等.我国几种植物植原体的快速分子鉴别与鉴定的研究[J].植物病理学报,2005,35(4)：293-299.
[108]王胜坤,王军,徐大平.四种桉树青枯菌DNA提取方法及PCR检测灵敏度比较[J]. Frest Pest and Disease Sept,2007,.5：4-7.
[109]邵雪玲,等.生物化学与分子生物实验指导.武汉大学出版社.2003：36-37
[110]Toth I. K., Hyman L. J. and Wood J. R.. A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants[J]. The Society for Applied Microbiology,1999,87, 158-166.
[111]王丽花,等.彩色马蹄莲欧文氏菌PCR检测[J].植物保护,2006,32(6)：75-78.
[112]魏景超.真菌鉴定手册[M].上海科学技术出版社,1979.
[113]Groves A. B.. A study of the sooty blotch disease of apples and the causal fungus Gloeodes pomigena. Va. Agric. Exp. Stn. Bull.,1933,50:1-43.
[114]Heuer H., Krsek M., Baker P., et al. Analysis of Actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients[J]. Applied and Environmental Microbiology,1997,63:3233-3241.
[115]Gomes N. C., Fagbola O., Costa R., et al. Dynamics of fungal communities in bulk and maize rhizosphere soil in the tropics[J]. Applied and Environmental Microbiology,2003..69:3758-3766.
[116]赵勇,李武,周志华,等.应用PCR-RFLP及PCR-TGGE技术监测农田土壤微生物短期动态变化[J1.南京农业大学学报,2005,28(3)：53-57.
[117]李延茂,胡江春,张晶,等.杉木连栽土壤微生物多样性的比较研究[J].应用生态学报,2005,16(7)：1276-1278.
[118]宋亚娜,包兴国,李隆等.利用DGGE法研究不同种植体系中根际微生物群落结构[J].生物学杂志,2006,23(5)：12-15,4.
[119]周琳,张晓君,李国勋,等.DGGE/TGGE技术在土壤微生物分子生态学研究中的应用[J].生物技术通报,2006,5：67-71.
[120]唐影.转基因抗虫棉对根区土壤细菌多样性影响的初步研究[D].中国农业科学院,2007.
[121]罗海峰,齐鸿雁,薛凯等.PCR-DGGE技术在农田土壤微生物多样性研究中的应用[J].生态学报,2003,23(8)：1570-1575.
[122]Teske A., Wawer C., Muyzer G. Distribution of sulfate-reducing bacteria in a stratified fjord(Mariager Fjord,Denmark)as evaluated by most-probable-number counts and denaturing gradient gel electro-phoresis of PCR-amplified ribosomal DNA fragments[J]. Applied and Environmental Microbiology,1996,62:1405-1415.
[123]Ferris M. J., Muyzer G, Ward D. M.. Denaturing gradient gel electrophor-esis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community[J]. Applied and Environmental Microbiology,1996, 62(2):340-346.
[1241李沁元,崔晓龙,张东华,等.云南腾冲热海两热泉菌藻席细菌多样性的研究[J].微生物学报,2004,44(4)：431-435.
[125]王涛,柴丽红,崔晓龙,等.免培养法对一热泉细菌多样性的初步研究[J].微生物学报,2003,43(5)：541-546.
[126]Eichner C. A., Erb R. W., Timmis K. N., et al. Thermal gradient gel electrophoresis analysis of bioprotection from pollutant shocks in the activated sludge microbial community[J]. Applied and Environmental Microbiology,1999, 65:102-109.
[127]Tsai Y. L., Olson B. H.. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction[J]. Applied and Environmental Microbiology,1992,58:2292-2295.
[128]Sekiguchi H., Watanabe M., Nakahara T. Succession of bacterial community structure along the Changjiang River determined by denaturing gradient gel electrophoresis and clone library analysis[J]. Applied and Environmental
Microbiology,2002,68(10):5142-5150.
[129]向少能,陈琳,何晓丽,等.水体微生物多样性PCR-DGGE分析方法的比较研究[J].重庆工学院学报(自然科学版),2007,7：74-78,97.
[130]Zoetendal E. G, Akkermans A. D., De Vos W. M.. Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria[J]. Applied and Environmental Microbiology,1998,64(10):3854-3859.
[131]Simpson J. M., Mccracken V. J., White B. A.. Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota[J]. Journal of Microbiological Methods,1999,36(3):167-179.
[132]卓凤萍.贡嘎蝠蛾幼虫肠道细菌多样性分析[D].重庆大学,2005.
[133]庞小燕,张宝让,魏桂芳等.应用TGGE技术分析人肠道中双歧杆菌的组成[J].微生物学报,2005,45(5)：738-743.
[134]Vainio E. J. and Hantula J.. Direct analysis of wood-inhabiting fungi using denaturinggradient gel electrophoresis of amplified ribosomal DNA[J]. Mycological Research,2000,104 (8):927-936.
[135]龙良鲲,羊宋贞,姚青,等.AM真菌DNA的提取与PCR-DGGE分析[J].菌物学报,2005,24(4)：564-569.
[136]沈洪,陈明杰,赵永昌,等.羊肚菌内生细菌DGGE鉴定[J].上海农业学报,2008,24(2)：58-60.
[137]梁新乐,朱扬玲,蒋予箭,等.PCR-DGGE法研究泡菜中微生物群落结构的多样性[J].中国食品学报,2008,8(3)：133-137.
[138]工海燕,张晓君,徐岩,等.浓香型和芝麻香型白酒酒酷中微生物菌群的研究[J].酿酒科技,2008,2：86-89,91.
[139]雷娟利,周艳,丁桔,等.不同蔬菜连作对土壤细菌DNA分子水多态性形响的研究[J].中国农业科学,2005,38(10)：2076-2083.
[140]王蒙.长江口九段沙湿地盐沼植物根围细菌群落结构和多样性的研究[D].上海,复旦大学,2006,52.
[141]Schabereiter-Gurtner C., Pinar G, Lubitz W., et al. Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18S rDNA sequence analysis[J]. J Microbiol Methods,2001,47:345-354.
[142]蔡莹.对虾养殖沉积环境细菌多样性的分子分析与若干可培养技术优化探讨[D].厦门,厦门大学,200
[143]原贵生,刘惠娟,刘贤谦等.七种药剂对枣缩果病病原菌—细交链孢菌抑制作用[J].2007,26(6)：28-30.
[144]刘惠娟,刘随存,刘贤谦,等.枣缩果病与果实生理指标关系的研究[J].山西林业科技,2007,1：19-21,32.
[145]屈建华.枣缩果病的克星—靓枣[J].果农园地,2009,1：48.
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