Adverse effect of the chitinolytic enzyme PjCHI‐1 in transgenic tomato on egg mass production and embryonic development of Meloidogyne incognita

A novel chitinase gene (PjCHI‐1) isolated from Paecilomyces javanicus, a non‐nematophagous fungus, and driven by a CaMV35S promoter, was delivered into CLN2468D, a heat‐tolerant cultivar of tomato (Solanum lycopersicum). T₁ tomato plants exhibited high endochitinase activity and reduced numbers of eggs and egg masses when infected with the root‐knot nematode (RKN) Meloidogyne incognita. The eggs found in transgenic tomato had lower shell chitin contents than eggs collected from control plants. Egg masses from transgenic plants exhibited higher chitinase activity than those from control plants. Moreover, only 30% of eggs from transgenic plants were able to develop to the multi‐cell/J1 stage, compared with more than 96% from control plants. The present study demonstrated that the expression of the PjCHI‐1 chitinase gene can effectively reduce the production of egg masses and repress the embryonic development of M. incognita, presenting the possibility of a novel agro‐biotechnological strategy for preventing crop damage by RKN.     

Root-specific expression of defensin in transgenic tobacco results in enhanced resistance against <Emphasis Type="Italic">Phytophthora parasitica</Emphasis> var. <Emphasis Type="Italic">nicotianae</Emphasis>

Phytophthora species are soil-borne pathogens that damage plants in both agro- and natural ecosystems. To suppress the devastating pathogen, we generated a root-specific expression system using a specific promoter (pPRP3) conferring elevated expression of the target gene in roots that are very susceptible to soil-borne pathogens. To verify root-specific expression, we compared β-glucuronidase (GUS) expression driven by a constitutive or root-specific promoters in shoots and roots. In histochemical and fluorometric assays, GUS activity was detected in whole tobacco plants when GUS expression was driven by p35S, but was detected only in the roots by pPRP3. We then expressed a pepper defensin (J1–1) gene in tobacco to elucidate its effect on plant resistance. The accumulation of J1–1 was also tissue-specific in transgenic tobacco plants. Finally, transgenic plants carrying GUS or J1–1 genes in combination with p35S or pPRP3 were inoculated with Phytophthora parasitica var. nicotianae and Pythium aphanidermatum. Disease symptoms were significantly suppressed in transgenic plants that accumulated J1–1, regardless of the promoter used. Furthermore, the expression of PR genes was induced in J1–1 transgenic plants, exhibiting much higher levels in p35S-driven J1–1 plants than in pPRP3::J1–1 plants. These results demonstrated that J1–1 transgenic plants were primed for enhanced expression of PR genes, which provided synergistic effects with the defensin for disease resistance.     

Plasmodiophora brassicae -induced expression of pyk20, an Arabidopsis thaliana gene with glutamine-rich domain

The expression of the pyk20 gene of Arabidopsis thaliana, which encodes a protein containing a glutamine-rich domain is up-regulated during Plasmodiophora brassicae infection. Transgenic A. thaliana plants harbouring a β-glucuronidase (uid A) reporter gene under the control of the pyk20 gene promoter were grown in soil and infected with P. brassicae resting spores. GUS expression in non-infected plants was found in stipules, apical meristem, leaf vascular tissues, vascular tissue of roots and in the root tips. After infection with P. brassicae, GUS staining was observed in the root hairs during primary infection and in galls in roots and hypocotyls during secondary infection phase. GUS expression during primary infection was also detected in a small number (approx. 0.01%) of zoosporangia. Sections of the GUS-stained galls showed reporter gene expression in infected and non-infected tissues. Northern-blot analysis using a pyk20 cDNA clone as a probe confirmed responsiveness of the pyk20 gene to P. brassicae infection.     

Synthetic tetramer of a Phytophthora sojae-inducible fragment from soybean GmaSKTI36 promoter improves its pathogen induction activities

Using pathogen-induced promoters to control expression of the functional genes in transgenic plants may greatly increase the chances of boosting disease resistance. However, the number of the inducible promoters is limited. Here, we found that soybean GmaSKTI36 gene is strongly induced upon Phytophthora sojae infection. Functional analysis showed that its promoter could mediate rapid and strong induction of GUS expression upon pathogen infection in both Nicotiana benthamiana leaves and soybean hairy roots. Then, a 122 bp fragment that was critical to the activity was successfully identified by a progressive 5′ deletion analysis. Importantly, we found that a synthetic promoter by tetramerizing this fragment could confer strong P. sojae induction activities. Overall, the results suggested that the GmaSKTI36 promoter, the 122 bp fragment, and the synthetic promoter are potentially useful pathogen-inducible promoters.     

含有多个应答逆境元件的合成启动子在水稻中的表达分析

 存在于启动子中的各种顺式作用元件在基因应答逆境的过程中起重要的作用，对这些元件进行适当组合是构建诱导型合成启动子的重要可行途径之一。本研究利用多个逆境应答元件(S盒、D盒、Gst1盒、TCA盒和LURP1基因启动子的-85~-46区)和CaMV35S核心(-46-+8)序列相连，组成一个人工合成启动子PRBS，并构建了GUS为报告基因的转化载体，通过农杆菌介导的遗传转化方法获得其水稻转基因植株。利用T₁代转基因水稻株系，通过检测GUS酶活性，研究了PRBS在不同逆境及外源激素作用下的表达特征。结果发现PRBS组成型表达水平低，可不同程度地应答稻瘟病病原菌侵染，机械损伤和外源激素(ABA、SA和MeJA)处理，表明PRBS可作为诱导型启动子用于水稻抗逆基因工程遗传改良。     

三种化合物处理对南方根结线虫生长发育及致病能力的影响

为明确不同浓度NH4HCO3、Cu SO4·5H2O和Fe Cl3·6H2O对南方根结线虫Meloidogyne incognita 2龄幼虫活性的影响,在盆栽条件下将化合物处理后的2龄幼虫接种番茄,通过根染色和根系冰冻切片法观察2龄幼虫的发育状况和根系内巨细胞的形成,并测定了番茄根系各生理指标的变化。结果表明,50mmol/L NH4HCO3、1.4mmol/L Cu SO4·5H2O和1.4 mmol/L Fe Cl3·6H2O处理南方根结线虫后,番茄根系内2龄、3龄及4龄幼虫数量均显著小于对照;根系内丙二醛、可溶性糖和游离氨基酸的含量均显著低于对照,根系活力显著增强;经NH4HCO3处理后,根系内巨细胞形成缓慢,14 d时出现巨型细胞空洞现象。3种化合物处理对南方根结线虫生长发育的抑制作用显著,处理后的线虫对番茄植株致病力减弱,表现为NH4HCO3抑制作用最强,Cu SO4·5H2O次之,Fe Cl3·6H2O最弱。     

Expression of δ-endotoxin Cry1EC from an inducible promoter confers insect protection in peanut (Arachis hypogaea L.) plants

BACKGROUND: Spodoptera litura (F.) is a polyphagous foliage insect and a major pest on peanut (Arachis hypogaea L.). Constitutive expression of δ‐endotoxin Cry1EC gives protection against S. litura, as reported earlier. In this study, insect bites and salicylic acid induced high‐level expression of Cry1EC was achieved in peanut. In order to achieve this, the expression of pathogenesis responsive promoter PR‐1a was enhanced by placing it downstream of the CaMV35S promoter in the pCAMBIA 1300 backbone. The resultant promoter CaMV35S(r)PR‐1a expressed a high level of insecticidal δ‐endotoxin Cry1EC. The Gus expression under the control of CaMV35S(r)PR‐1a served as a convenient marker for evaluation of promoter response to different treatments. RESULTS: Transgenic events that showed a very low level of uninduced expression and no expression in seeds were selected. The Cry1EC expression in leaves increased nearly eightfold in the selected event, following induction by salicylic acid. Both the salicylic‐acid‐treated and the S. litura‐bitten leaves showed the highest expression after 2 days. Leaves from salicylic‐acid‐induced transgenic plants caused 100% mortality of S. litura at all stages of larval development. CONCLUSION: The results suggest that high expression of inducible promoters provides a good strategy for the development of safer transgenic food and feed crops. Copyright © 2010 Society of Chemical Industry     

Infection and colonization of peanut pods by Aspergillus parasiticus and the expression of the aflatoxin biosynthetic gene,nor-1, in infection hyphae

The aflatoxigenic fungi, Aspergillus flavus and A. parasiticus infect a wide variety of crops, all of which produce oil-rich seed. A histological study of the host–pathogen interaction between peanut,Arachis hyphogea , and A. parasiticus was performed in a system where peanuts remained attached to the plant and were inoculated without wounding. For infection studies, a genetically-tagged strain of A. parasiticus, G5, was engineered to harbor the β-glucuronidase (GUS) reporter gene under control of the nor-1 promoter from the aflatoxin biosynthetic pathway. There was a similar temporal pattern of aflatoxin B1 production and appearance of GUS activity in cultures ofA. parasiticus G5. This strain was used to follow infection and aflatoxin production during colonization of undamaged, drought-stressed peanuts. The fungus colonized all tissues of the peanut pod and appeared to gain ingress through the corky layer of the pericarp. Both intra- and inter-cellular colonization were observed. Fungal colonization of the cotyledons resulted in visible depletion of storage bodies within cells. Two morphologically distinct types of hyphae, wider hyphae and narrower hyphae, were seen throughout the pod tissues. Statistical analysis revealed that the narrower hyphae were significantly more likely to produce GUS activity than wider ones. GUS activity was found in hyphae infecting the pericarp, embryo and cotyledons indicating expression of aflatoxin biosynthetic genes in these tissues. Interestingly, GUS activity was not observed in the hyphae colonizing the testa.     

In-plant activation of root-specific expression of a cytotoxic gene disrupts the development of the root-knot nematode,Meloidogyne javanica

Plant hosts can be engineered to disrupt the development of sedentary plant-parasitic nematodes or proper functioning of the feeding sites the nematodes induce. The use of constitutive promoters to express dsRNAs or nematode inhibitor proteins may be unreliable because of possible silencing or yield penalty from continuous expression in a plant host. This ill-effect can be avoided if a root-specific, nematode-responsive promoter (NRP) is used to drive the target nematode-inhibitory message. This study used the In Plant Activation (INPACT) system to express a barstar-controlled barnase in galls of Meloidogyne javanica and assessed how the engineered tobacco lines affected the growth and development of the nematodes. Of the 11 combinations of four NRPs and the CaMV 35S promoter assessed, the AtCel1 and TobRB7 combinations activated specific expression of split β-glucuronidase (GUS) and barnase genes in and around giant cells. The same NRP combination directed expression of the barnase gene in tobacco roots also constitutively expressing the barstar gene (SPBB transgenic lines). On roots of six T₁ SPBB lines, there was up to 94% reduction in the number of galls with significantly smaller adult females compared to those on wild-type plants. Some of the females on lines SPBB4-1 and SPBB-4-2, for example, were not associated with galls. The results indicated the engineered plants disrupted M. javanica development and demonstrate the potential for controlled and localized expression of peptides, such as those that could block specific effectors, to disrupt initiation, formation, establishment, or proper functioning of feeding cells induced by damaging sedentary nematodes.     

Suitability of weed species prevailing in Spanish vineyards as hosts for root-knot nematodes

Commercial vineyards in southern Spain were surveyed and sampled during October to December 2004 to determine the extent to which common weeds present were suitable hosts of root-knot nematodes infesting soils of those vineyards. Seven weed species commonly growing in grapevine soils in southern Spain were found infected by either Meloidogyne incognita or M. javanica: Amaranthus retroflexus (redroot pigweed), Anchusa azurea (ox-tongue), Chenopodium album (goosefoot), Erodium moschatum (musk stork’s bill), Malva rotundifolia (low mallow), Sinapis alba (white mustard), and Solanum nigrum (black nightshade). The host suitability of the weeds to root-knot nematodes was evaluated on the basis of root galling severity and nematode population densities in soil and roots. Also, the host–parasite relationship in these naturally Meloidogyne-infected weeds was examined. All the weed species in the study were considered suitable hosts for M. incognita and M. javanica because: (a) high Meloidogyne spp. populations occurred in roots and surrounding soil of the weed species; (b) the severity of root galling was high, and (c) well-established permanent feeding sites were observed in the histopathological studies of infected root tissues. In addition, this study presents the first reports of S. alba and A. azurea as hosts for M. incognita, and of E. moschatum as a new host for M. javanica, thus increasing the list of reported weed hosts for Meloidogyne spp. These results indicate that noticeable population densities of M. incognita and M. javanica can be maintained or increased in these weeds, at population levels higher than those previously reported for the same nematodes infecting grapevine roots. The weeds infesting vineyards thus represent an important source of inoculum of Meloidogyne spp., and furthermore may act as reservoirs of these nematodes which can be disseminated within or among vineyards by agricultural operations.     

Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.     

Tight regulation of allene oxide synthase (AOS) and allene oxide cyclase-3 (AOC3) promote <Emphasis Type="Italic">Arabidopsis</Emphasis> susceptibility to the root-knot nematode <Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

Biosynthesis of the oxylipin jasmonic acid (JA) in Arabidopsis thaliana is catalyzed by a single allene oxide synthase (AOS)-encoding gene and four genes encoding four functional allene oxide cyclase (AOC) polypeptides (AOC1, AOC2, AOC3, and AOC4). To elucidate the biological activities of the JA pathway in regulating the plant defense response to plant-parasitic nematodes, transgenic lines carrying the GUS reporter gene under the control of individual AOC or AOS promoters were examined. Upon penetration by second-stage juveniles (J2 s), promoter activities of AOC1, AOC3 and AOC4 appeared in the root tip and root-elongation zone, with AOC3 demonstrating highest induction. At 5 days AOC3 activity continued to be highly pronounced in the stele and root cortex, associated with nematode invasion throughout gall initiation and maturation. AOS expression appeared 3 days postinfection and accompanied all later infection stages. Mutant lines were analyzed: disruption in AOS rendered plants more resistant to nematode infection, as reflected by the decreased number of females produced on this line; loss-of-function of AOC3 rendered plants more susceptible to nematode infection. Oxylipins derived from the 9- and 13-lipoxygenase pathways were assayed for their direct inhibitory activity toward M. javanica J2 s. Clear nematicidal activity of the bioactive 9- and 13-hydroperoxides was observed. Oxylipins produced by divinyl ether synthase, colneleic acid, colnelenic acid and ω5(Z)-etherolenic acid demonstrated strong inhibitory activity. These data, along with those of other assayed oxylipins, suggest that temporal and spatial fine tuning of the JA route allowing nematodes parasitism on plant host.     

Nematicidal fluorescent pseudomonads for the in vitro and in vivo suppression of root knot (Meloidogyne incognita) of Capsicum annuum L

BACKGROUND: Considerable attention has been paid to plant‐growth‐promoting rhizobacteria (PGPR), especially the fluorescent group of Pseudomonas species, as the best alternatives to chemicals for facilitating ecofriendly biological control of soil‐ and seedborne microorganisms. On the basis of their novel plant‐growth‐promoting attributes, two rhizobacteria Pseudomonas aeruginosa VP1 and VP2 selected out of over 63 isolates from the rhizosphere of chilli (Capsicum annuum) were identified as potential candidates for biocontrol of the root‐knot nematode Meloidogyne incognita on chilli. RESULTS: The nematicidal activity of both strains was evaluated in vitro and in vivo for their efficacy against M. incognita. P. aeruginosa VP2 exhibited strong nematicidal activity in comparison with VP1, based on the in vitro killing of the second‐stage juveniles (J2) of M. incognita. Seed bacterisation with both strains VP1 and VP2 was able to manage root‐knot M. incognita on chilli (C. annuum) in a pot trial study. Increase in root and shoot length and in fresh and dry weight of root and shoot and reduction in the root‐knot index over the control were attained. In overall performance, VP2 was 29.5% more effective than VP1, and about 30% more effective than the control (non‐bacterised). CONCLUSION: The application of P. aeruginosa VP1 and P. aeruginosa VP2 controls the development of M. incognita in C. annuum, and hence they are recommended as efficient plant growth promotors and biocontrolling agents for raising healthy crop of C. annuum that can promote the growth of plants and reduce the nematode (M. incognita) population. Copyright © 2012 Society of Chemical Industry     

和田鞘氨醇杆菌AMCC 100218菌株的筛选、鉴定及杀线虫特性

为筛选对南方根结线虫具有致死效果的生防细菌,从山东省10个县市蔬菜主产区番茄根际土壤中分离可培养细菌,采用离体杀线虫试验测定分离菌株对南方根结线虫Meloidogyne incognita的致死活性,结合生理生化特征及分子生物学方法对高效杀线虫菌株进行分类鉴定,同时对其杀线虫特性进行表征,并通过盆栽试验进一步验证其生防潜力。结果显示,从山东省蔬菜主产区番茄根际土中分离到1株高效杀线虫菌株AMCC 100218,结合生理生化试验与16S rRNA序列分析,鉴定此菌株为和田鞘氨醇杆菌Sphingobacterium hotanense;该菌株对南方根结线虫2龄幼虫的致死效果可达88.87%,其杀线虫活性物质具有较好的热稳定性和储存稳定性,且耐碱不耐酸;盆栽试验结果表明,该菌能够显著减少土壤中的虫口密度,降低番茄发病率。表明和田鞘氨醇杆菌AMCC 100218菌株是1株具有防治根结线虫病潜力的生防细菌。     

The early nodulin enod40 is induced by,and also promotes arbuscular mycorrhizal root colonization

Expression of Tenod40, a tobacco (Nicotiana tabacum cv. Xanthi nc) homologue of Msenod40, a gene expressed early during alfalfa (Medicago sativa) nodule development, was significantly and consistently increased (about three-fold) in tobacco roots of plants grown in sand and colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices, as compared to non-mycorrhizal controls. In alfalfa (Medicago sativa cv. Gilboa), a similar induction of Msenod40 expression was also detected in mycorrhizal vs. non-mycorrhizal roots. In the same experimental system, the application of the cytokinin 6-benzylaminopurine to non-mycorrhizal roots resulted in a similar increase in Msenod40 expression. A significantly higher incidence of AM fungal colonization (vesicles per cm of root) in M. sativa cv. Gilboa roots was observed following application of luteolin-induced Sinorhizobium meliloti exudates as compared to plants amended with non-induced exudates. Moreover, overexpression of the Mtenod40 gene in Medicago truncatula plants under the control of a 35S constitutive promoter resulted in significantly enhanced colonization as compared to non-transgenic plants. The fact that enod40 gene expression was increased both in a legume and in a non-legume during colonization by the AM fungus, and that colonization levels were higher in enod40 -overexpressing transgenic plants strongly suggests that this gene is involved in the establishment of the fungal partner in its host.     

The Use of a GUS Transformant of Trichoderma Harzianum, Strain T3a, to Study Metabolic Activity in the Spermosphere and Rhizosphere Related to Biocontrol of Pythium Damping-off And Root Rot

The activity of Trichoderma harzianum in the spermosphere and rhizosphere of different plant species was studied by use of a beta-glucuronidase (GUS) transformant (strain T3a). Hereby, direct observation of micro-habitats supporting metabolic activity of T. harzianum is reported. Germination of conidia and mycelial growth were not supported by exudates from healthy roots of various ages. Instead, growth and activity of T. harzianum depended on access to dead organic substrates such as seed coats, decaying roots, and wounds, including those caused by infecting pathogens. A correlation between the GUS activity of T. harzianum and the biomass of Pythium ultimum in infected roots was established. On the basis of our observations, we suggest that the biocontrol ability of T. harzianum involves competition with the pathogen for substrates including the seed coat, and wounded or infected root tissue.     

Penetration and reproduction of root-knot nematodes on cucurbit species

Cucurbits are often cultivated in rotation with Solanaceae in double-cropping systems. Most cucurbits have been described as susceptible to root-knot nematodes (RKN) but little is known on their relative levels of susceptibility. Because RKN species differ in rates of root invasion and reproductive traits, isolates of M. arenaria, M. incognita and M. javanica were compared on five cucurbit hosts in experiments run in a climate growth chamber. They included zucchini squash cv Amalthee, cucumber cv Dasher II, melon cv Pistolero, pumpkin cv Totanera and watermelon cv Sugar Baby. All cucurbits were susceptible to the three RKN isolates although M. javanica showed higher invasion rates, faster development and higher egg production than M. arenaria on the selected cucurbits. Apparent differences among cucurbits were primarily due to root invasion rates and formation of egg masses. Both Cucumis species (cucumber and melon) were better hosts for nematode invasion and reproduction than zucchini squash, followed by watermelon. Large invasion rates followed by small reproduction traits were linked to M. incognita on zucchini squash. Reduced invasion rates and egg mass formation along with delayed early development were shown on watermelon.