Suppression of Meloidogyne incognita by the endophytic fungus Acremonium implicatum from tomato root galls

Specific endophytes with biocontrol potential might occur in diseased plant tissues. We isolated an endophytic fungus from tomato root galls infected with Meloidogyne incognita and identified it as Acremonium implicatum based on morphology and internal transcribed spacer sequences. Its biocontrol potential was tested in vitro and in pot and field experiments. In the in vitro test, 96.0% of second-stage juveniles of M. incognita were killed by a culture filtrate of A. implicatum after 48 h. The fungus also suppressed egg hatching, with only 36.3% of treated eggs hatching compared with 87.3% of control eggs. Pot experiments showed that A. implicatum inhibited the formation of root galls, with 40.6 galls per treated plant compared with 121.6 on control plants. A. implicatum reduced the nematode population in soil, with 151.1 nematodes per 100 g treated soil and 375.1 in control soil. Field experiments demonstrated that the root gall index of treated plants (25) was markedly lower than that of control plants (96). In conclusion, A. implicatum has excellent potential for the biocontrol of M. incognita.     

Host-delivered RNAi-mediated root-knot nematode resistance in <Emphasis Type="Italic">Arabidopsis</Emphasis> by targeting <Emphasis Type="Italic">splicing factor</Emphasis> and <Emphasis Type="Italic">integrase</Emphasis> genes

Root-knot nematodes (RKNs) are one of the most important biotic factors limiting crop productivity in many crop plants. The major RKN control strategies include development of resistant cultivars, application of nematicides and crop rotation, but each has its own limitations. In recent years, RNA interference (RNAi) has become a powerful approach for developing nematode resistance. The two housekeeping genes, splicing factor and integrase, of Meloidogyne incognita were targeted for engineering nematode resistance using a host-delivered RNAi (HD-RNAi) approach. Splicing factor and integrase genes are essential for nematode development as they are involved in RNA metabolism. Stable homozygous transgenic Arabidopsis lines expressing dsRNA for both genes were generated. In RNAi lines of splicing factor gene, the number of galls, females and egg masses was reduced by 71.4, 74.5 and 86.6%, respectively, as compared with the empty vector controls. Similarly, in RNAi lines of the integrase gene, the number of galls, females and egg masses was reduced up to 59.5, 66.8 and 63.4%, respectively, compared with the empty vector controls. Expression analysis revealed a reduction in mRNA abundance of both targeted genes in female nematodes feeding on transgenic plants expressing dsRNA constructs. The silencing of housekeeping genes in the nematodes through HD-RNAi significantly reduced root-knot nematode infectivity and suggests that they will be useful in developing RKN resistance in crop plants.     

Nematicidal potential of Artemisia annua and its main metabolites

Nematotoxic effect of an aqueous extract of Artemisia annua and its components caffeic acid, chlorogenic acid (5-caffeoylquinic acid, 5-CQA), artemisinin and the related semi-synthetic artesunate, was investigated on the root-knot nematode Meloidogyne incognita and the potato cyst nematode Globodera rostochiensis and on the virus-vector dagger nematode Xiphinema index. Juveniles of M. incognita and G. rostochiensis and females of X. index were exposed to 500, 250 and 125 μl ml^?1 solutions of the A. annua aqueous extract, caffeic acid, chlorogenic acid and artesunate and to 50 μl ml^?1 solution of artemisinin for 2, 4, 8 and 24 h. Egg masses of M. incognita and cysts of G. rostochiensis were exposed for 24, 48, 96 h and 1 or 2 weeks only to the extract solutions. Aqueous extract was highly effective on G. rostochiensis juveniles, whereas M. incognita juveniles were affected only at long exposure times. Adversely, egg hatch inhibition was strong on M. incognita and poor or minimal on G. rostochiensis. Females of X. index were sensitive only to long exposures to the highest extract concentration. Both caffeic and chlorogenic acid did not affect juveniles of M. incognita but were highly active on G. rostochiensis juveniles and X. index females even at the lowest concentration. Artesunate toxicity was almost zero on M. incognita and low on X. index females, but high on G. rostochiensis juveniles. Artemisinin solution was lethal to more than 50 % of G. rostochiensis juveniles within 24 h, but did not affect M. incognita juveniles and X. index females. Results suggest different roles of the tested compounds in the biocidal activity on each target nematode species. The extract of A. annua and its main phytochemicals seem to have a potential to be developed into new nematicidal formulates, though their activity should be validated in the soil.     

Resistance to root-knot nematodes on passion fruit genotypes in Brazil

With the expansion of passion fruit cultivation in Brazil, phytosanitary problems have increased, among them, the occurrence of root-knot nematodes. This research aimed to study the response of passion fruit genotypes to Meloidogyne incognita, M. javanica and M. enterolobii in addition to evaluating the life cycle of M. enterolobii in the passion fruit genotype ‘FB 200’. The genotype response was carried out in a greenhouse. Each pot’s soil was inoculated with 5000 eggs. Gall index, egg mass index and nematode reproduction factors were evaluated at 120 days after inoculation. All genotypes studied were resistant to M. incognita, M. javanica and M. enterolobii, except ‘Roxinho do Kênia’, which was susceptible to the three nematode species. The life cycle of M. enterolobii in “FB 200” passion fruit was studied in a growth chamber at 26 °C with a photoperiod of 12 h. Seven days after transplantation, each plant was inoculated with approximately 400 second-stage juveniles. Evaluations were done at 7, 14, 21, 28, 35, 42 and 49 days post inoculation. The nematode did not complete its life cycle.     

Efficacy of a formulated product containing <Emphasis Type="Italic">Quillaja saponaria</Emphasis> plant extracts for the control of root-knot nematodes

The nematicidal effect of a formulated product containing extract from Quillaja saponaria was evaluated against the root-knot nematodes. The product QL Agri® 35 (QL) was tested to record the effect on second stage juveniles motility, egg hatch and also against field populations in greenhouse experiments contacted in three different locations of Greece. Convulsive movement of second stage juveniles of Meloidogyne incognita was recorded after exposure for 8 days at a series of doses, while the most paralyzed juveniles were counted at the dose of 8 mg l^?1. There was also a gradual decrease in the number of juveniles emerging from egg masses of the same nematode species when the dose of Q. saponaria was increased from 0 to 8 mg l^?1. In greenhouse experiments, the use of Q. saponaria could control root-knot nematodes and prevent nematodes increase in soil. The present study demonstrates that the use of Q. saponaria extract has the ability to control root-knot nematodes. Control given by Q. saponaria in field populations infecting cucumber was similar to that of cadusafos (Rugby®) and oxamyl (Vydate®) under the tested dosages and the specific conditions of the experiments.     

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.     

Toxicity of fungal endophyte secondary metabolites to plant parasitic nematodes and soil-borne plant pathogenic fungi

Fungi isolated from the cortical tissue of surface sterilized tomato roots collected from field plots produced secondary metabolites in nutrition broth that were highly toxic toMeloidogyne incognita. Especially strains ofFusarium oxysporum were highly active with 13 of 15 strains producing culture filtrates toxic to nematodes. The mechanism of action of the toxic metabolites produced by the non-pathogenicF. oxysporum strain 162 with proven biological control ofM. incognita in pot experiments was investigated. These metabolites reducedM. incognita mobility within 10 min of exposure. After 60 min, 98% of juveniles were inactivated. Juveniles were initially inactivated within a few minutes of exposure, but with exposure of 5 h 50% of the juveniles were dead and 24 h exposure resulted in 100% mortality. In a bioassay with lettuce seedlings metabolite concentrations > 100 mg/l reduced the number ofM. incognita juveniles on the roots comparing to the water control. TheF. oxysporum toxins were highly effective towards sedentary parasites and less effective towards migratory endoparasites. Nonparasitic nematodes were not influenced at all. Metabolites of strain 162 also reduced significantly the growth ofPhytophthora cactorum, Pythium ultimum andRhizoctonia solani in vitro. Secondary metabolites of endophytic fungi on plant-parasitic nematodes and soil-borne fungi should be considered for control of plant parasitic nematodes and plant pathogenic fungi. The results also show the need for proper selection of target nematodes inin vitro bioassays.     

Thermal time requirements of root-knot nematodes on zucchini-squash and population dynamics with associated yield losses on spring and autumn cropping cycles

The present research was undertaken to evaluate the effects of soil temperature on the life cycle of root-knot nematodes (RKN) on zucchini-squash in growth chambers and to assess the relationship between Meloidogyne incognita soil population densities at planting (Pi), its multiplication rate, and crop losses of zucchini in field conditions. Thermal requirements for M. incognita and M. javanica were determined by cultivating zucchini plants in pots inoculated with 200 second stage juveniles (J2) of each Meloidogyne species at constant temperatures of 17, 21, 25, and 28 °C. Number of days from nematode inoculation until appearance of egg laying females and until egg hatching were separately recorded. For life cycle completion, base temperatures (Tb) of 12?ºC and 10.8?ºC and accumulated degree-days above Tb (S) of 456 and 526, were estimated for M. incognita and M. javanica, respectively. The relationship between fruit weight and M. incognita Pi fits the Seinhorst damage function, but differed accordingly to the cropping season, spring or autumn. Tolerance limits for M. incognita on zucchini were 8.1 J2 per 250 cm³ of soil in spring and 1.5 in autumn cropping cycles, and the minimum relative yields were 0.61 in spring and 0.69 in autumn. Zucchini-squash was a poorer host for M. incognita in spring than in autumn, since maximum multiplication rates (a) and equilibrium densities (E) were lower in spring (a?=?16–96; E?=?274–484) than in autumn (a?=?270–2307; E?=?787–1227).     

In-vitro and in-vivo nematicidal activities of the cyclic dodecapeptide omphalotin A

Omphalotin A, a cyclic dodecapeptide produced by submerged cultures of the basidiomycete Omphalotus olearius, exhibited in-vitro and in-vivo nematicidal activity. Meloidogyne incognita was the most sensitive nematode. At 2.0 mg litre⁻¹, 50% of the nematodes were dead after one hour. Heterodera schachtii, Radopholus similis and Pratylenchus penetrans were affected at higher concentrations. Incorporated into agar, the compound prevented infection of cucumber seedlings by M. incognita at concentrations of 1 mg litre⁻¹ and higher. In glasshouse tests, complete protection of cucumbers and lettuce was achieved between 2.5 and 10 mg litre⁻¹. No insecticidal activity was observed when Plutella xylostella, Phaedon cochleariae or Spodoptera frugiperda were fed material containing 4 g kg⁻¹ of omphalotin A. © 1999 Society of Chemical Industry     

Influence of different fertilization regimes on plant-parasitic nematodes in the black soil region of Northeast China

Plant-parasitic nematodes can cause serious plant diseases and adversely affect crop production. We investigated the temporal and vertical dynamics of plant-parasitic nematodes in northeast China to determine if long-term fertilizer application suppresses the population of plant-parasitic nematodes and influences their vertical distribution in the black soil region. The long-term fertilizer application lasted for 14 years, and included three treatments: pig manure combined with chemical fertilizer (MCF), chemical fertilizer (urea and ammonium phosphate, CF), and no fertilizer (NF). A 3-year corn-wheat-soybean rotation was grown on the field site, soil samples were obtained in the corn phase and nematodes were extracted, identified and counted. Six plant-parasitic nematode genera with relative abundance over 0.1% were found in this study. Heterodera was the dominant genus in all three fertilizer treatments, and its relative abundance was highest in NF (45.4%) and lowest in MCF (32.8%). Fertilizer application had a significant effect on abundance of total soil nematodes but not on either plant-parasitic nematodes or Heterodera. Significant differences in the abundance of total soil nematodes, plant-parasitic nematodes and Heterodera were observed among soil depths and interaction of fertilizer × soil depth. Total soil nematodes were mainly distributed in the 0–20 cm soil layer, and plant-parasitic nematodes and Heterodera were mostly distributed in the 10–30 cm soil layer. Fertilizer application did not show a significant effect on plant-parasitic nematodes or Heterodera at any of the sampling depths from 0 to 80 cm. The results indicated that fertilizer has no effective control on plant-parasitic nematodes when dominant genera and their respective plant hosts exist in relatively high abundance.     

南方根结线虫Col基因分子克隆及其VIGS效应分析

为了明确线虫角质层胶原蛋白基因(Col)与根结线虫病害的关系,利用从线虫基因组中预测的Col设计特异引物,克隆了南方根结线虫Meloidogyne incognitacol基因MiCol.该基因完整编码区长903bp,编码300个氨基酸.克隆的MiCol与预测的MiCol基因序列一致性高达99.67％,氨基酸序列一致性高达100％.利用病毒介导的基因沉默技术,将其导入番茄植株并接种南方根结线虫,60 d后,沉默载体pTV-MiColi处理番茄植株的根结数比空载体对照及清水对照分别减少49.4％和49.2％,表明MiCol基因沉默显著降低了南方根结线虫的侵染数量.     

Soil application of imidacloprid and related SAR-inducing compounds produces effective and persistent control of citrus canker

Soil application of the systemic insecticide imidacloprid (Admire®, Bayer Crop Science) produced season-long control of citrus canker caused by Xanthomonas citri sbsp. citri. Imidacloprid is a neo-nicotinoid that breaks down in planta into 6-chloronicotinic acid, a compound closely related to the systemic acquired resistance (SAR) inducer isonicotinic acid. Potted Swingle citrumelo seedlings (Citrus paradisi × Poncirus trifoliata) were treated with imidacloprid and the SAR inducers, isonicotinic acid, and acibenzolar-s-methyl as soil drenches or with acibenzolar-s-methyl as a foliar spray 1week prior to inoculation of immature leaves with X. citri sbsp. citri. Seedlings were re-inoculated four times over a 24-week period. SAR induction was confirmed by expression of the PR-2 gene (β-1,3 glucanase). Soil drenches of imidacloprid, isonicotinic acid, and acibenzolar-s-methyl induced a high and persistent up-regulation of PR-2 gene expression and reduced the number of canker lesions for up to 24 weeks compared to 4 weeks for foliar acibenzolar-s-methyl. Soil applied inducers of SAR reduced canker lesions up to 70% compared with the untreated inoculated plants. Lesions on leaves were small, necrotic, and flat compared to pustular lesions on inoculated untreated plants. Populations of X. citri sbsp. citri per leaf were reduced 1–3 log units in soil-treated plants compared to inoculated untreated plants.     

Baseline sensitivity of Monilia yunnanensis to the DMI fungicides tebuconazole and triadimefon

Monilia yunnanensis was recently identified as a new species causing brown rot of peach in China. Sterol 14α-demethylase inhibitors (DMIs) continue to be important in the management of brown rot of Monilinia spp. worldwide. Tebuconazole and triadimefon are two kinds of DMI fungicides that may be used for brown rot control in China. To establish the baseline sensitivity of M. yunnanensis to these two DMI fungicides, 203 M. yunnanensis single spore isolates were collected. Measurements of sensitivity to the two fungicides were based on inhibition of mycelial growth. For both fungicides, the sensitivity distribution was a unimodal curve, with an EC₅₀ range (the effective concentration to inhibit mycelial growth by 50 %) of 0.0001–0.0644 μg/ml for tebuconazole and 0.2311–1.7477 μg/ml for triadimefon. The M. yunnanensis isolates were obtained from orchards where DMI fungicides have not been used for peach brown rot control, thus the fungicide sensitivity distribution established in this study can be considered as the baseline for monitoring the resistance development in M. yunnanensis once the DMI fungicides are used to control peach brown rot.     

An antibiotic fusaricidin: a cyclic depsipeptide from Paenibacillus polymyxa E681 induces systemic resistance against Phytophthora blight of red-pepper

In this study fusaricidin, a cyclic depsipeptide isolated from Paenibacillus polymyxa E681 (E681), was demonstrated to control Phytophthora blight infection caused by Phytophthora capsici in red-pepper. The minimal inhibitory concentration (MIC) of fusaricidin was found to be 16 ppm against P. capsici. The disease severity of P. capsici was 40% at 0.1 ppm of fusaricidin when compared with water-treated control (81.7%) on post-treatment, whereas the disease severities on pre-treatment were 45% and 83.3% in fusaricidin (0.1 ppm) and water-treated control, respectively, in red-pepper plants. Significant (P?<?0.05) disease suppression was observed on treatment with fusaricidin (0.1 ppm) by foliar spray and soil drench. The disease severity was drastically reduced to 3.3% by soil drench of fusaricidin (1.0 ppm), whereas in water-treated control, the disease severity was 83.3% in the first experiment. Fusaricidin at 0.1 ppm reduced disease severity of P. capsici to 27.5% when compared with positive control (43.1%) and water-treated control (66.2%) in the second experiment. Soft rot disease in tobacco was suppressed upon treatment with fusaricidin at 1.0 ppm by leaf infiltration. RT-PCR analyses of Arabidopsis thaliana revealed that there was an up-regulation of pathogenesis-related (PR) gene expression in wild type A. thaliana (Col-0), while there was no accumulation of PR genes, which implies that the mechanism of protection might be based on a salicylic acid-dependent pathway. This is the first report that fusaricidin exhibits protection against plant pathogens in addition to activity as an antibiotic agent. Hence, E681 can play a role in plant protection by secretion of ISR elicitors including fusaricidin.     

Does soil warming affect the interaction between Pasteuria penetrans and Meloidogyne javanica in tomato plants?

A system to grow tomato plants infected by Meloidogyne javanica under constant temperatures of 18, 21, 24, 27 and 30 °C was developed and used to assess how temperature and the application of the biological control bacterium Pasteuria penetrans affected plant growth, the nematode population and endospore production. Each plant was inoculated with 300 second‐stage juveniles (J2) with four or five spores of P. penetrans attached to their cuticles or with 300 nematodes without P. penetrans. Increasing soil temperature increased tomato growth, the number of endospores per female, and the number of galls of M. javanica at the end of 38 days. Increasing temperatures up to 27 °C also increased the number of egg masses produced by M. javanica. Presence of P. penetrans reduced the numbers of galls and egg masses at all temperatures by up to 52.2% and 61.4% at 27 and 30 °C, respectively. Pasteuria penetrans reduced the M. javanica population even at soil temperatures of 18 and 21 °C. However, temperatures of 27 and 30 °C enhanced nematode control and the production of P. penetrans endospores is faster. The system developed in this work is simple and efficient for growing plants under constant temperatures and can be used for different purposes.     

Biological characterization and genetic diversity analysis of two species of Pestalotiopsis causing twig dieback of Myrica rubra

Chinese bayberry (Myrica rubra Siebold & Zucc) is an evergreen fruit tree with high ecological and economic values in China. In recent years, a new twig dieback disease caused by Pestalotiopsis mangiferae and P. vismiae was observed in major M. rubra-producing areas of Zhejiang and Fujian Provinces, causing serious economic losses. In this study, 16 isolates of P. mangiferae and 27 isolates of P. vismiae were obtained from diseased leaves, roots and branches of M. rubra from different regions. The optimum growth temperature of the two species of Pestalotiopsis was determined to be 20–25 °C, while the optimum temperature for the germination of conidia was 25–35 °C. The two species of Pestalotiopsis showed rich genetic diversity. Inoculating the conidial suspension of one or both of the two species of the Pestalotiopsis on detached leaves or branches of M. rubra could cause lesions surrounding the inoculation sites with the frequency of 100 %. Moreover, necrotic lesions could be observed on inoculated potted plants with the frequencies of 33.3 % for P. mangiferae, 25 % for P. vismiae and 50 % for a mixed inoculum.     

黄瓜幼苗内生真菌诱导植株对南方根结线虫的抗性

在2007-2008年的一项温室测验中,10株黄瓜幼苗内生真菌表现出了对南方根结线虫较好的防效,为了阐明其作用方式,用裂根试验设计研究了10个菌株诱导黄瓜产生根结线虫抗性的能力.同一植株的根系被分为彼此隔离的挑战根系和应答根系,镰刀菌Fusarium spp.菌株Fu234和Fu654、毛壳菌Chaetomium sp.菌株Ch1001、叶点霉Phyllosticta sp.菌株Ph511在挑战根系中接种后,应答根系中的线虫入侵总数显著降低,较对照分别降低了42.4％、35.7％、38.4％、23.6％(第1次测定)和63.6％、45.2％、51.0％、37.0％(重复测定),这4个菌株诱导了黄瓜对根结线虫入侵的抗性.同时,Fu234、Fu654、Ch1001和拟青霉Paecilomyces sp.菌株Pa972接种后应答根系中的卵与雌虫的比值显著减少,相比对照其比值分别降低了24.3％、37.7％、48.3％、21.2％(第1次测定)和21.6％、39.8％、30.0％、46.2％(重复测定),这4个菌株诱导了黄瓜对根结线虫繁殖的抗性.     

Effect of abamectin on root-knot nematodes and tomato yield

BACKGROUND: Tomato growers in Shandong Province, China, commonly face heavy root‐knot nematode infestations. Current methods of control include cadusafos and methyl bromide (MeBr), but alternative methods are required because of the high toxicity of these pesticides and the ecological risk of their use. Therefore, abamectin soil applications were evaluated for their potential to control soil nematodes in a series of laboratory tests, greenhouse pot experiments and field trials. RESULTS: Laboratory tests showed that abamectin exhibited rapid knockdown of Meloidogyne incognita, with LC₅₀ and LC₉₀ values that were superior to those of cadusafos and averaged 7.06 and 21.81 mg L^?1. In the greenhouse pot experiment, soil applications of abamectin provided significant M. incognita control similar to that provided by cadusafos while maintaining excellent plant height and vigour. In the field trials, abamectin exhibited excellent control effects to nematodes while giving a higher tomato yield. There was a 19.3–39.0% yield increase from the various treatments compared with the control, and the best results were obtained from the highest dose of abamectin. CONCLUSION: The results of this study demonstrated that abamectin has the potential to be used as an effective alternative to MeBr and cadusafos for nematode control in tomato production in Shandong Province. Copyright © 2012 Society of Chemical Industry