Histological and proteomics analysis of apple defense responses to the development of Colletotrichum gloeosporioides on leaves

Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. is an important fungal pathogen known to cause glomerella leaf spot (GLS). The objective of this study was to analyze the infection of C. gloeosporioides on leaves of apple cv. Fuji (resistant) and cv. Gala (susceptible) and apply proteomics techniques to study the apple defense responses 48 h after inoculation (h.a.i.). On both of cultivars, C. gloeosporioides started to germinate at 3 h.a.i. on adaxial surface and produced appressoria adhering to epidermal cell juxtapositions. Histological analysis showed more stratified parenchyma in leaves of cv. Fuji than cv. Gala associated with differences in the chemical composition of cell walls. Total and unique proteins expressed by cvs. Fuji and Gala at 3, 12, 24 and 48 h.a.i. were detected by comparative proteomes analysis. A total of 42 unique proteins expressed at 24 and 48 h.a.i. were identified by MALDI/TOF mass spectrometry, and most of these proteins were identified as directly involved in defense responses.     

Investigation and genetic mapping of a Glomerella leaf spot resistance locus in apple

Apple Glomerella leaf spot (GLS) is a severe fungal disease that damages apple leaves during the summer in China. Breeding new apple varieties that are resistant to the disease is considered the best way of controlling GLS. Fine mapping and tightly linked marker are critically essential for the preselection of resistant seedlings. In this study, a population of 207 F₁ individuals derived from a cross between ‘Golden Delicious’ and ‘Fuji’ was used to construct a fine simple sequence repeat (SSR)‐based genetic linkage map. The position of R_gls, a locus responsible for resistance to GLS, was identified on apple linkage group (LG) 15 using SSR markers CH05g05 and CH01d08, which was adapted from a published set of 300 SSR markers that were developed using the bulked segregant analysis (BSA) method. These two SSR markers flanked the gene, and its recombination rate was 8.7% and 23.2%, respectively. A total of 276 newly developed SSR markers around the target region and designed from the genome apple assembly contig of LG15 were screened. Only nine of these were determined to be linked to the R_gls locus. Thus, a total of 11 SSR markers were in linkage with R_gls, and mapped at distances ranging from 0.5 to 33.8 cM. The closest marker to the R_gls locus was S0405127, which showed a genetic distance of approximately 0.5 cM. The first mapping of the gene R_gls was constructed, and the locations of the 11 effective primers in the ‘Golden Delicious’ apple genome sequence were anchored. This result facilitates better understanding of the molecular mechanisms underlying the trait of resistance to GLS and could be used in improving the breeding efficiency of GLS‐resistant apple varieties.     

炭疽叶枯病菌诱导的不同苹果种质中防御酶活性及丙二醛含量比较

以苹果品种‘秦冠’、‘富士’及二者的杂交种‘QF-2’为试材,室内接种炭疽叶枯病菌,发现前者表现为感病,后二者均表现为抗病.接种后的0~4 d内,寄主叶片内的SOD、POD、CAT活性均出现先升高后降低的趋势,在接种1 d后出现峰值,且在抗病种质上明显高于感病种质.PPO活性在接种后也是呈现先上升后下降的趋势,但感病种质'秦冠'的上升速度较快,峰值出现较早.对三份种质来说,受病原菌的诱导,MDA含量均在接种1 d后达到高峰,然后缓慢下降,但其含量变化与种质间的抗性水平差异没有明显的相关性.     

有机硅助剂在苹果炭疽叶枯病化学防控中的减药增效作用评价

有机硅助剂具有低毒、易降解的特性,可提高农药的药液展布性和渗透性。本研究分别采用室内离体叶片法和田间试验评价了2种有机硅助剂对吡唑醚菌酯和代森锰锌防治苹果炭疽叶枯病的减药增效作用。室内离体试验结果表明,杀菌剂浓度减半的处理以及模拟雨水冲刷处理均导致吡唑醚菌酯和代森锰锌的防效显著下降;而添加了有机硅助剂后,2种处理的防效显著提高,并且恢复到与正常施药量相同的水平。田间试验结果表明,正常情况下杀菌剂浓度减半后防效显著下降;而施用有机硅助剂后,浓度减半的吡唑醚菌酯和代森锰锌的防效显著高于不施用有机硅的处理。本研究结果表明,施用有机硅助剂可以在减少化学农药实际用量的情况下,保持较高的防效,这对于化学农药的减施增效具有重要意义。     

披针叶黄华抗杉木炭疽菌活性物质研究

预试验中发现披针叶黄华地上部分乙醇浸提物具有抗杉木炭疽菌活性 ,进一步用不同溶剂 ,在不同 p H值下萃取得到 4个组分 .生测表明披针叶黄华中生物碱部分对杉木炭疽菌的抑菌活性最强 .其生物碱对杉木炭疽菌分生孢子萌发较对菌丝生长的抑制活性强 ,对杉木炭疽菌的分生孢子和菌丝的 EC50分别为 1 36.0 μg/m L和 1 555.0 μg/m L     

Analysis of a Secreted Aspartic Peptidase Disruption Mutant of Glomerella cingulata

Peptidases have been implicated in the pathogenicity of fungi that cause disease in plants. Expression of the secreted aspartic peptidase gene (gcsap), of a Glomerella cingulata isolate pathogenic on apples, is induced during appressorium formation. To determine whether the secreted aspartic peptidase (GcSAP) is essential to pathogenicity, gcsap was disrupted using a vector containing a 637 bp fragment of genomic DNA that encodes the sequence spanning the two active site aspartic acid (Asp) residues. To ensure that the truncated gcsap gene products could not have residual peptidase activity the codons for the active site residues Asp¹¹² and Asp²⁹⁷ were both mutated to histidine residues. Both PCR and Southern analysis confirmed disruption of gcsap. Neither gcsap mRNA nor GcSAP activity was detected in the disruption mutant. Pathogenicity tests on fruit from three apple cultivars showed that GcSAP was not required for pathogenicity. The disruption mutant grew on medium containing protein as the sole source of nitrogen because G. cingulata secretes a previously undetected peptidase(s). A serine peptidase that had a pH optimum between pH 7.0 and 8.0 and a K _m of 0.25 mM for the synthetic substrate succinyl-Ala–Ala–Pro–Phe-p-nitroanilide was identified.     

苹果贮藏期间炭疽病菌侵染过程的初步研究

将苹果炭疽病菌配成不同浓度孢子悬浮液接种至处于贮藏期的苹果上 ,在 2 0℃恒温下暗光培养 ,对病菌侵染过程进行研究。结果表明 :对于针刺接种秦冠苹果 ,病斑接种成功率随接种孢子浓度增大而增大 ,当接种浓度在 1× 10 5个孢子 /ml以上时 ,接种成功率为 10 0 % ;病菌潜伏期和潜育期与接种浓度呈显著负相关 ;潜育期至潜伏期所需的有效积温和产孢时病斑直径在不同浓度接种间差异不显著 ,其值分别为 99.2℃和 19.6mm ;病斑扩展速率在接种浓度 1× 10 4 ～ 1× 10 5个孢子 /ml范围内最大。     

锥栗炭疽病的研究 Ⅰ.病原的确定及其生物学特性

通过分离培养,人工接种和病原鉴定,证实锥栗炭疽病是由围小丛壳菌[Glomerellacingulata(Stonem.)Spauld.etSchrenk]侵染所致,其无性世代为胶孢炭疽菌(ColletotrichumgloeosporioidesPenz).病原菌菌落在PDA加锥栗叶煎汁培养基上生长较好.菌丝生长最适温度为25～30℃,最适pH值为6.0.pH3～11均能产生分生孢子,pH8.5时产生分生孢子的能力最强.在自由水条件下分生孢子才有较高的萌发率(39.2%).分生孢子萌发pH值范围为2～10,最佳为6.5;锥栗叶煎汁培养基对孢子萌发有明显的促进作用.     

解淀粉芽胞杆菌TS-1203对苹果炭疽叶枯病菌的抗生作用

为明确解淀粉芽胞杆菌TS-1203对苹果炭疽叶枯病菌的抗生作用, 采用平板对峙法和菌丝生长速率法以及孢子萌发法测定了解淀粉芽胞杆菌TS-1203对苹果炭疽叶枯病菌菌落生长和孢子萌发的抑制作用?结果表明:菌株TS-1203对苹果炭疽叶枯病菌具有显著的抑制作用, 其菌液对病菌的抑制率高达70.4%; 其发酵液对苹果炭疽叶枯病菌的抑制率随着发酵液浓度的增加而升高, 尤其当发酵液浓度为15%时, 其抑菌率为 59.0%; 发酵液处理苹果炭疽叶枯病菌孢子24 h后, 其萌发抑制率与芽管生长抑制率分别为78.2%与64.3%, 且芽管出现畸形; 利用TS-1203发酵液处理苹果离体叶片后接种炭疽叶枯病菌, 其防效为70.1%?综合分析表明, 解淀粉芽胞杆菌TS-1203对苹果炭疽叶枯病菌具有良好的抑制作用?     

套袋对富士苹果轮纹病和炭疽病的影响

本试验通过不同时期套袋的方法,研究了富士苹果轮纹病及炭疽病的侵染规律。结果表明:富士苹果自谢花后7～11d即开始感染轮纹病,花后23～27d为侵染高峰;而炭疽病菌的侵染高峰期为花后27～43d。适期套袋（花后7d）可有效防治这两种病害的为害。