Involvement of ethylene signaling in Azospirillum sp. B510-induced disease resistance in rice

A bacterial endophyte Azospirillum sp. B510 induces systemic disease resistance in the host without accompanying defense-related gene expression. To elucidate molecular mechanism of this induced systemic resistance (ISR), involvement of ethylene (ET) was examined using OsEIN2-knockdown mutant rice. Rice blast inoculation assay and gene expression analysis indicated that ET signaling is required for endophyte-mediated ISR in rice.

Abbreviations: ACC: 1-aminocyclopropane-1-carboxylic acid; EIN2: ethylene-insensitive protein 2; ET: ethylene; ISR: induced systemic resistance; JA: jasmonic acid; RNAi: RNA interference; SA: salicylic acid; SAR: systemic acquired resistance     

Effects of Colonization of a Bacterial Endophyte,Azospirillum sp. B510, on Disease Resistance in Rice

Agriculturally important grasses contain numerous diazotrophic bacteria, the interactions of which are speculated to have some other benefits to the host plants. In this study, we analyzed the effects of a bacterial endophyte, Azospirillum sp. B510, on disease resistance in host rice plants. Rice plants (Oryza sativa cv. Nipponbare) were inoculated with B510 exhibited enhanced resistance against diseases caused by the virulent rice blast fungus Magnaporthe oryzae and by the virulent bacterial pathogen Xanthomonas oryzae. In the rice plants, neither salicylic acid (SA) accumulation nor expression of pathogenesis-related (PR) genes was induced by interaction with this bacterium, except for slight induction of PBZ1. These results indicate the possibility that strain B510 is able to induce disease resistance in rice by activating a novel type of resistance mechanism independent of SA-mediated defense signaling.     

Complete Genomic Structure of the Cultivated Rice Endophyte Azospirillum sp. B510

We determined the nucleotide sequence of the entire genome of a diazotrophic endophyte, Azospirillum sp. B510. Strain B510 is an endophytic bacterium isolated from stems of rice plants (Oryza sativa cv. Nipponbare). The genome of B510 consisted of a single chromosome (3 311 395 bp) and six plasmids, designated as pAB510a (1 455 109 bp), pAB510b (723 779 bp), pAB510c (681 723 bp), pAB510d (628 837 bp), pAB510e (537 299 bp), and pAB510f (261 596 bp). The chromosome bears 2893 potential protein-encoding genes, two sets of rRNA gene clusters (rrns), and 45 tRNA genes representing 37 tRNA species. The genomes of the six plasmids contained a total of 3416 protein-encoding genes, seven sets of rrns, and 34 tRNAs representing 19 tRNA species. Eight genes for plasmid-specific tRNA species are located on either pAB510a or pAB510d. Two out of eight genomic islands are inserted in the plasmids, pAB510b and pAB510e, and one of the islands is inserted into trnfM-CAU in the rrn located on pAB510e. Genes other than the nif gene cluster that are involved in N₂ fixation and are homologues of Bradyrhizobium japonicum USDA110 include fixABCX, fixNOQP, fixHIS, fixG, and fixLJK. Three putative plant hormone-related genes encoding tryptophan 2-monooxytenase (iaaM) and indole-3-acetaldehyde hydrolase (iaaH), which are involved in IAA biosynthesis, and ACC deaminase (acdS), which reduces ethylene levels, were identified. Multiple gene-clusters for tripartite ATP-independent periplasmic-transport systems and a diverse set of malic enzymes were identified, suggesting that B510 utilizes C₄-dicarboxylate during its symbiotic relationship with the host plant.     

两种砧木对樱桃番茄青枯病抗性及根际微生物数量的影响

青枯病是危害我国华南地区番茄生产最严重的土传性病害之一。为探讨在人工接种青枯菌条件下,两种不同砧木嫁接对番茄青枯病的抗性及根际微生物数量的影响。该研究分别用砧木“番砧1号”和“茄砧21号”与樱桃番茄“粉贝贝”进行嫁接,采用稀释平板法对各嫁接组合的根际微生物进行分离。结果表明:(1)采用抗病砧木嫁接显著提高了番茄的抗病性,降低了发病率和病情指数,延缓了发病时间。(2)青枯菌在侵染植株过程中呈现动态变化,其数量从根系到地上茎部逐渐减少;砧穗嫁接植株、砧木自根嫁接植株根际基质和根系中的青枯菌数量降低,且地上茎部中的病原菌数量显著低于接穗自根嫁接植株;接种青枯菌后,各嫁接组合根际基质中的细菌、真菌和放线菌的数量随发病期的变化呈先增加后降低的趋势。(3)采用抗病砧木嫁接总体上提高了植株根际微生物总量、细菌及放线菌的数量,降低了真菌的数量,改善了嫁接植株根际微环境,对降低番茄青枯病发病率具有重要作用。     

黑麦草-内生真菌共生体对磷缺乏的生理生态反应

选取感染和未感染的黑麦草为材料,在田间盆栽条件下研究内生真菌感染对宿主植物抵抗磷胁迫方面的贡献。结果表明,土壤中缺磷或内生真菌感染对黑麦草地上部生长的影响不显著,但内生真菌感染对植株地下部生长和生理指标有明显影响。缺磷条件下,内生真菌感染有助于黑麦草地下部分的生长,表现在根系总长度更长,生物量更大;同时根中酚类物质和有机酸的含量也显著高于未感染植株,但因酚类物质和有机酸总量增加的同时并未伴随着二者浓度的增加,由此推测,内生真菌在改变宿主黑麦草根系代谢活动方面的贡献有限。此外,内生真菌感染显著提高了宿主植物的磷利用效率,这可能和缺磷条件下内生真菌感染植株具有更高的酸性磷酸酶活性有关。     

内生真菌感染对宿主羊草抗病性的影响

以感染内生真菌的天然禾草羊草为实验材料,通过体外纯培养条件下的内生真菌、感染内生真菌的离体叶片和在体叶片对3种病原菌的抑菌实验,以探讨内生真菌对宿主植物羊草在抗病性方面的贡献。结果表明:体外纯培养条件下,分离自羊草的内生真菌Epichlobromicola对新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)和枝孢霉(Cladosporium sp.)这3种病原菌都具有抑制作用,抑菌率分别达56.22%,46.93%和45.15%,且内生真菌培养滤液可以有效抑制这3种病原菌的孢子萌发,平均萌发率分别为30.4%,15.7%和16.4%;宿主植物叶片在离体条件下,内生真菌感染可以有效降低羊草叶片受C.lunata和C.sp.侵染后的病斑数或病斑长度,但对B.sorokiniana不起作用,甚至提高了叶片的病斑数及病斑长度,而离体叶片提取液对不同病原菌均有不同程度的抑制作用;在体条件下,内生真菌均可以通过降低叶片病斑数来增强羊草植株对这3种病原菌的抗性。由此看来,内生真菌E.bromicola对宿主植物羊草在抗病原菌侵染方面有一定的增益作用。     

Assessment of genetic variation in tomato （Solanum lycopersicum L.） inbred lines using SSR molecular markers

A study was conducted to determine the genetic diversity of 39 determinate and indeterminate tomato inbred lines collected from China, Japan, S. Korea, and USA. Using 35 SSR polymorphic markers, a total of 150 alleles were found with moderate levels of diversity, and a high number of unique alleles existing in these tomato lines. The mean number of alleles per locus was 4.3 and the average polymorphism information content (PIC) was 0.31. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering at genetic similarity value of 0.85 grouped the inbred lines into four groups, where one USA cultivar formed a separate and more distant cluster. The most similar inbred lines are from USA, both with determinate type, whereas the most different lines are from USA (Us-16) and Japan (Ja-2) with determinate and indeterminate growth habit, respectively. Clustering was consistent with the known information regarding geographical location and growth habit. The genetic distance information reported in this study might be used by breeders when planning future crosses among these inbred lines.     

干旱胁迫下内生真菌感染对黑麦草叶内几种同工酶的影响

以内生真菌感染(endophyte-infected,EI)与不感染(endophyte-free,EF)的黑麦草(Lolium perenne L．)种子建立实验种群,分别对其施加长时间不同强度的干旱胁迫,通过比较黑麦草体内过氧化物酶(POD)、超氧化物歧化酶(SOD)、多酚氧化酶(PPO)活性及其同工酶谱的变化以探讨保护酶系统在内生真菌——植物共生体的抗旱性方面所作的贡献。研究结果表明,水分胁迫和内生真菌对黑麦草3种酶的影响不仅表现在总量上而且表现在同工酶的酶谱及各区带的酶活力上。就总酶活力而言,EI和EF植株中POD、SOD和PPO的活性均随着干旱胁迫强度的增加而增加,进一步将EI和EF植株的酶活力进行比较,发现与EF植株相比,EI植株中POD和PPO的活性相对较低,而SOD的活性相对较高。从同工酶的谱带数量和强弱来看,POD同工酶各区带活力均随干旱胁迫强度的增加而增加,EI植株叶片增加的幅度高于EF叶片,而且EI叶片在重度胁迫下出现了1条新带SOD同工酶各区带活力在EI叶片中有随干旱胁迫增加而增加的趋势,而在EF叶片中有些区带酶活力增强,有些区带酶活力减弱,且EI叶片在中度胁迫下出现了1条新带;PPO同工酶随干旱胁迫的增强,EI和EF叶片均表现为有些区带酶活力增强,有些区带酶活力减弱。总之,内生真菌的感染虽然没有显著提高宿主植物黑麦草POD、SOD和PPO的活性,但使宿主黑麦草对干旱胁迫的反应更为迅速,其中既包括POD、SOD等酶活力的迅速升高,也包括新酶带的产生。     

Reaction of some tomato cultivars to tomato leaf curl virus and evaluation of the endophytic colonisation with Beauveria bassiana on the disease incidence and its vector,Bemisia tabaci

Tomato line LA1478 and Pusa Ruby were resistant to tomato leaf curl virus (TLCV) disease. They registered higher plant height, number of branches, total phenol content and yield per plant than the other cultivars. Variety Peto 86 was tolerant to the disease while the other popular tomato cultivars, i.e. Ace, Early Pack, Money Maker, Prichard and Strain B were highly susceptible to the disease. Plant height and number of branches per plant revealed significantly positive association with fruit yield per plant. The disease index of TLCV exhibited significant negative correlations with plant height, total phenol content and fruit yield per plant – 0–4 and 5–25 adult whiteflies were observed on resistant susceptible cultivars. In the case of epiphytically colonisation by Beauveria bassiana conidia, not all developing hyphae on the leaf surface penetrated the whitefly cuticle. Many of the germ tubes elongated to a short distance before terminating its growth. On the other hand, the rapid staining of tomato tissues injected with B. bassiana conidial suspension indicates that the entomopathogenic fungus was established inside tomato tissues until the end time of the trial. The direct injection with the spore suspension yielded high post-colonisation, where the fungus was recovered from sites distant from the point of inoculation. This indicates that the fungus has the potential to move throughout the plant tissues. Laboratory bioassay of tomato whitefly feeding on tomato tissues containing B. bassiana conidial spores indicates that plant endophytic colonisation with entomopathogenic fungi may reduce insect survival on these plants. LT₅₀ values of the test diet were between three and four days. The mortality of Bemisia tabaci was high in the case of endophytically colonisation compared to epiphytically one (90.0% compared to 10.0% during three days) for whiteflies fed tomato tissues containing 1.5 × 10⁷ B. bassiana spores/ml. Application of B. bassiana as an artificial endophyte inside tomato plants can be an important component in the integrated control of tomato whiteflies. The endophytic colonising can achieve biocontrol effect based on induced disease resistance in plant tissues. According the available references, this is the first report on B. tabaci controlling by plant endophytic treatment.     

内生真菌感染对宿主羽茅及邻生植物抗病性的影响

Epichloë内生真菌能够影响宿主植物的抗病性, 但目前关于内生真菌感染对宿主邻生植物抗病性影响的研究很少。该研究首先调查了内蒙古呼伦贝尔天然草地中植物病害发生情况, 发现在常见牧草中禾本科植物病害最为严重, 其中感染内生真菌的羽茅(Achnatherum sibiricum)的总病情指数最低。进一步比较了与羽茅邻生及非邻生植物的病害差异, 结果显示羽茅显著降低了邻生羊草(Leymus chinensis)、狼针草(Stipa baicalensis)和冰草(Agropyron cristatum)的褐斑病病情指数。为探究羽茅对邻生植物病害的影响是否与其内生真菌有关, 分别从内生真菌发酵液、离体叶片和植株层面进行了室内验证试验。结果表明: 羽茅的2种内生真菌Epichloë gansuensis和Epichloë sibiricum发酵液显著抑制了新月弯孢(Curvularia lunata)、根腐离蠕孢(Bipolaris sorokiniana)、核盘菌(Sclerotinia sclerotioru)和三叶草核盘菌(Sclerotinia trifoliorum)的生长; 对于离体叶片, E. gansuensis和E. sibiricum显著减小了宿主叶片受4种病原真菌侵染后的病斑长度。在新月弯孢和根腐离蠕孢的植株接种条件下, 内生真菌显著降低了宿主羽茅及邻生植物羊草的病害程度。该研究首次发现并验证了羽茅内生真菌对邻生禾草抵御褐斑病有一定的增益作用。     

Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.     

Identification of I‐7 expands the repertoire of genes for resistance to Fusarium wilt in tomato to three resistance gene classes

The tomato I‐3 and I‐7 genes confer resistance to Fusarium oxysporum f. sp. lycopersici (Fol) race 3 and were introgressed into the cultivated tomato, Solanum lycopersicum, from the wild relative Solanum pennellii. I‐3 has been identified previously on chromosome 7 and encodes an S‐receptor‐like kinase, but little is known about I‐7. Molecular markers have been developed for the marker‐assisted breeding of I‐3, but none are available for I‐7. We used an RNA‐seq and single nucleotide polymorphism (SNP) analysis approach to map I‐7 to a small introgression of S. pennellii DNA (c. 210 kb) on chromosome 8, and identified I‐7 as a gene encoding a leucine‐rich repeat receptor‐like protein (LRR‐RLP), thereby expanding the repertoire of resistance protein classes conferring resistance to Fol. Using an eds1 mutant of tomato, we showed that I‐7, like many other LRR‐RLPs conferring pathogen resistance in tomato, is EDS1 (Enhanced Disease Susceptibility 1) dependent. Using transgenic tomato plants carrying only the I‐7 gene for Fol resistance, we found that I‐7 also confers resistance to Fol races 1 and 2. Given that Fol race 1 carries Avr1, resistance to Fol race 1 indicates that I‐7‐mediated resistance, unlike I‐2‐ or I‐3‐mediated resistance, is not suppressed by Avr1. This suggests that Avr1 is not a general suppressor of Fol resistance in tomato, leading us to hypothesize that Avr1 may be acting against an EDS1‐independent pathway for resistance activation. The identification of I‐7 has allowed us to develop molecular markers for marker‐assisted breeding of both genes currently known to confer Fol race 3 resistance (I‐3 and I‐7). Given that I‐7‐mediated resistance is not suppressed by Avr1, I‐7 may be a useful addition to I‐3 in the tomato breeder's toolbox.     

土施糖类对丛枝菌根真菌侵染率和产孢的影响

丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)不能进行光合作用,需要宿主植物提供碳水化合物供其完成整个生命周期,添加外源物质调控AMF和宿主植物的关系被认为是一种可行的措施。通过盆栽实验种植番茄,探索土施不同糖类对摩西球囊霉Glomus mosseae的侵染率、产孢能力和功能(宿主植物生长和养分)的影响。结果表明,添加葡萄糖和蔗糖可提高接种了摩西球囊霉的番茄的地上部生物量以及磷、钾吸收量,但对地上部氮吸收量影响不显著;添加麦芽糖和淀粉对地上部生物量及氮磷钾养分吸收量的影响均不显著。添加糖类处理,土壤碱解氮均有下降趋势;土壤速效磷、速效钾随着地上部磷和钾吸收量增加有下降趋势。糖类添加对土壤有机质没有影响。添加不同糖类均提高了AMF的侵染率,其中添加蔗糖处理的侵染率较单独施用摩西球囊霉菌处理增加了114%。单独施用摩西球囊霉菌剂处理土壤孢子数为10个/g,添加葡萄糖和淀粉处理的孢子数均为8个/g,添加蔗糖和麦芽糖处理的孢子数均为11个/g,添加糖类均对AMF产孢无显著影响。