Functional evaluation of culture filtrates of Bacillus subtilis and Pseudomonas fluorescens on the mortality and hatching of Meloidogyne javanica

Rhizospheric bacteria Bacillus subtilis and Pseudomonas fluorescens are two widely tested biological control agents against root-knot nematodes (RKN) of different crops. However, their performance as bio-control agents varies with their place of origin. Culture filtrates of rhizospheric bacteria contain some intermediary metabolites that have nematicidal activity. An in vitro experiment was undertaken to evaluate the functionality of culture filtrates of B. subtilis (MN252542.1) and P. fluorescens (MN256394.1) at different concentrations (1.0%, 2.5%, 5.0%, 7.0%, 10.0% and 25.0%) on the hatching and mortality of Meloidogyne javanica at different time span. Bacterial strains were isolated from rhizospheric soils of Bangladesh. At three days after incubation (DAI), 25.0% concentration of culture filtrates of both B. subtilis and P. fluorescens showed 100.0% mortality of second stage juveniles (J₂) of M. javanica. Additionally, 25.0% concentration of culture filtrates of both bacteria showed 100.0% inhibition of hatching at one week after incubation (WAI). A decreasing trend in hatching of M. javanica was observed with the increment of the concentration of culture filtrates and progression of incubation time. The findings of this experiment reveal that culture filtrates of these accessions of B. subtilis and P. fluorescens are effective for controlling M. javanica and would be potential candidates for developing bio-nematicides.     

Phosphonate fertilizers suppressed root knot nematodes Meloidogyne javanica and M. incognita

The efficacy of the phosphonate fertilizers, Calphos^® (a.i. calcium phosphonate), Magphos^® (a.i. magnesium phosphonate and potassium phosphonate) and Phosphoros^® (a.i. potassium phosphonate) against two species of root knot nematodes (RKN), Meloidogyne javanica and M. incognita is evaluated. Laboratory experiments showed that Calphos^®, Magphos^® and their main components inhibited egg hatching and caused 100% mortality of the second stage juveniles (J2s) of the two RKN species; the hatching inhibition effects persisted after transferring the egg masses of both species to water. However, Phosphoros^® (0.5%) did not suppress egg hatching or the survival of J2s of both RKN species. No hatching occurred when egg masses were treated for one week with the nematicide Vydate L^® (2 ml/l), however, J2s hatched when the Vydate L^® treated egg masses were moved to water. The glasshouse study indicated that Magphos^®, Calphos^® and Phosphoros^® reduced root galling caused by M. javanica by 98, 66 and 47%, respectively, in comparison to the untreated controls. Magphos^® resulted in the lowest number of root galls formed by M. incognita, the reduction was 84%. In contrast, Calphos^® and Phosphoros^® reduced galling by 47 and 39%, respectively. The Magphos^® treatment resulted in the lowest numbers of egg masses and the lowest reproductive factor (RF) of both nematode species. However, plants treated with Phosphoros^® resulted in higher foliage weights compared with the application of the other two fertilizers and the untreated plants.     

Management of Fusarium oxysporum f. sp. lycopersici and root-knot nematode disease complex in tomato by use of antagonistic fungi,plant resistance and neem

Simultaneous infestation with root-knot nematodes (RKN) and Fusarium oxysporum f. sp. lycopersici (FOL) leads to formation of a disease complex that increases crop losses than effect of either RKN or FOL. In this study a management programme involving plant resistance, biological control agents, and neem was carried out to manage RKN and fusarium wilt disease complex. The biological control agents were Purpureocillium lilacinum (PL) and Trichoderma harzianum (TH) while the RKN was Meloidogyne javanica. In vitro dual culture plates were set up to test the interaction of biological control agents and FOL. Greenhouse experiments were conducted using two tomato cultivars Rambo F1 and Prostar F1. The treatments were; PL, TH, PL–TH, neem, PL neem, TH neem, and PL–TH neem. Each treatment was replicated four times and the treatments set up in a randomised complete block design in the greenhouse. Inhibition of FOL mycelial growth by TH and PL was 51.9%, and 44% respectively by the ninth day in vitro culture plates. In the cultivar, Prostar F1, the treatments PL–TH, PL, and TH in the presence or absence of neem had a FOL disease severity score significantly lower than the untreated control. Host resistance sufficed to prevent infection of Rambo F1 with FOL. The treatments PL–TH, PL and TH reduced FOL propagules and M. javanica juveniles in the roots and performed even better when combined with neem in both tomato cultivars. Therefore, a host that is resistant combined with biological control agents and organic amendments can be used in the management of RKN and FOL in tomato production.     

Comprehensive report on the prevalence of root-knot nematodes in the Poonch division of Azad Jammu and Kashmir,Pakistan

The present study documents the root-knot nematodes (RKN) fauna of the Poonch division in Azad Jammu and Kashmir infecting vegetables. An overall prevalence of 40% of RKN was recorded. Of the four districts investigated, maximum prevalence was recorded in district Poonch with 59%, followed by Sudhnuti with 58%. The lowest prevalence of RKN was found in districts Bagh (29%) and Haveli (33%). Out of 15 vegetables investigated, RKN was found on five crops. The highest prevalence of 37.8% was recorded on okra, followed by 31.3% on cucumber and 17.5% on tomato. RKN was less prevalent on eggplant (8.3%) and beans (7.7%). Three RKN species, that is Meloidogyne incognita, Meloidogyne javanica and Meloidogyne arenaria, were found infecting the hosts. M. javanica was found to be the most prevalent followed by M. incognita and M. arenaria. This trend was found in all the districts. Overall prevalence of M. javanica as sole population was 9% and that of M. incognita was 2%. Meloidogyne arenaria was not found in any of the fields as sole population. The prevalence of M. incognita with M. javanica or M. arenaria as mixed populations was 8% and 5%, respectively, and that of M. javanica with M. arenaria was 4%. Similarly, all the three species prevailed as mixed populations in 12% of the fields in the division. The severity of RKN infections, measured as galling index, was found to be variable within each infected field (GI 2–9). Identification of RKN species was based on the morphology of perineal patterns and confirmed by molecular SCAR and CO1 makers based identification. In conclusion, RKN were distributed in the Poonch division and M. javanica was predominant. Cucumber, okra, tomato and eggplant were severely attacked by these nematodes warranting the adoption of stringent control strategies for their management.     

Evaluation of burdock and Mountain almond leaf extracts against Meloidogyne javanica on tomato

Abstract

Root-knot nematodes (Meloidogyne spp.) are one of the most harmful plant pathogenic nematodes worldwide. Application of some herbal products can safely reduce negative effect of these nematodes. In the present study, the effect of aqueous extracts of Amygdalus scoparia and Arctium lappa on hatching and mortality of second-stage juveniles of M. javanica evaluated under laboratory condition and LC₃₀, LC₅₀, LC₇₀ and LC₉₀ values were determined by probit analysis from March to November 2016. Tomato seeds (cv. Early-Urbana) were sown in 1.5?kg plastic pots and simultaneously were inoculated with 4000 eggs and second stage juveniles (J2s) of M. javanica and soil-drenched (50?ml/pot) with selected concentrations of A. scoparia viz. 0.37, 0.54, 0.8 and 1.39% and A. lappa viz. 0.51, 0.85, 1.4 and 2.91%. The experiments were carried out in completely randomized design tests with four replications. Plant growth parameters as well as nematode population indices were calculated 60?days after inoculation. Results showed that after 120?hours, leaf extracts of A. scoparia at the rate of 7.5 and 10%, and leaf extract of A. lappa at the rate of 10% lead to 100% inhibition of M. javanica egg hatching under laboratory condition. Leaf extracts of both of the tested plants at the rate of 2% caused 100% mortality of J2s. Any increase in concentration of used plant extracts significantly improved the growth indices in both of the inoculated and uninoculated tomato plants. As compared to control, application of A. scoparia leaf extract at the rate of 2%, reduced the number of galls, egg masses and eggs per root system as well as the number of J2s per pot and reproduction factor of nematode by 37, 43, 45, 73 and 46%, and in the case of A. lappa, these indices reduced by15, 26, 27, 74 and 28%, respectively. Our results showed potential of leaf extracts of A. scoparia and A. lappa for management of M. javanica infecting tomato plants.     

贝莱斯芽孢杆菌TMQ-KSL-1分离鉴定及其发酵液对番茄根结线虫的生防作用

镰刀菌(Fusarium spp.)和根结线虫(Meloidogyne spp.)都是植物的重要病原物,这两种病原物在寄主植物中存在着非常复杂的互作关系,可导致严重的植物土传病害。为探寻对番茄根结线虫病害具有高效防治作用的优良菌株,本研究以禾谷镰刀菌(Fusarium graminearum)为靶标病菌,采用平板稀释涂布法从多年种植番茄的设施大棚土壤中分离和筛选到一株抑菌效果较好的生防细菌菌株TMQ-KSL-1,根据形态特征、生理生化特性和16S rRNA基因测序对该菌株进行鉴定;测定不同浓度的发酵液及发酵上清液对根结线虫卵孵化率以及根结线虫二龄幼虫死亡率的影响,通过盆栽实验分析其发酵液对根结线虫病害的防治效果。结果表明,菌株TMQ-KSL-1具有较强的杀线虫活性,其发酵液和发酵上清液处理48 h线虫卵孵化抑制率分别为94.76%和90.72%;处理24 h番茄根结线虫二龄幼虫的校正死亡率分别为100%和97.37%;菌株TMQ-KSL-1发酵液100倍稀释液、200倍稀释液对番茄根结线虫病害防治效果分别为59.54%和12.14%,且100倍液处理防效与阿维菌素500倍液处理防效(6...     

Effect of Phenamiphos on Heterodera schachtii and Meloidogyne javanica

Aqueous solutions of technical-grade phenamiphos [ethyl 3-methyl-4-(methylthio) phenyl (1-methylethyl) phosphoratnidale] were used in hatching chambers to test, under laboratory tory conditions, the effect of phenamiphos on the hatching and movement of Meloiclogyne javanica and Heterodera schachtii. Hatch of M. javanica and H. schachtii eggs was depressed 70 and 88% by nematicide at 0.48 and 4.80 μg/ml, respectively. The infectivity of second-stage larvae of both species was affected by concentrations as low as 0.01 μg/ml. At least 0.5 μg/ml was required to decrease the movement of larvae of M. javanica and H. schachtii. To decrease the movement of H. schachtii males toward females, 10 μg/ml was required. In a field experiment using a 15% granular formulation, 5 kg/ha a.i. significantly reduced infection of sugarbeet roots by H. schachtii.     

Effects of Pseudomonas fluorescens strain UTPF5 on the mobility,mortality and hatching of root-knot nematode Meloidogyne javanica

The root-knot nematode Meloidogyne spp. includes important plant pathogens worldwide. This study has considered nematode Meloidogyne javanica second stage larvae activity in the extracts of Pseudomonas fluorescens strains UTPF5 and cytotoxic effect of the strain on the nematode. The movement of second stage larvae of nematodes in water agar medium at four concentrations of bacterial extracts and second stage larvae mortality rate of hatching nematode and bacterial strains in vitro were affected. Different concentrates of the strain UTPF5 effect nematode larvae movement and disposal of the same. Bacterial extraction kills almost 100% of the larvae hatching after 24?h and a complete ban on egg hatch of biocontrol nematodes and nematode indicated that root-knot nematode larvae movement on the right attract the bacteria P. fluorescens to extract in the first place.     

Enhanced Nematicidal Activity of Organic and Inorganic Ammonia-Releasing Amendments by Azadirachta indica Extracts

The nematicidal activities of ammonium sulfate, chicken litter and chitin, alone or in combination with neem (Azadirachta indica) extracts were tested against Meloidogyne javanica. Soil application of these amendments or the neem extracts alone did not reduce the root galling index of tomato plants or did so only slightly, but application of the amendments in combination with the neem extracts reduced root galling significantly. Soil analysis indicated that the neem extract inhibited the nitrification of the ammonium released from the amendments and extended the persistence of the ammonium concentrations in the soil. In microplot experiments, tomato plants were grown in pots filled with soils from the treated microplots. The galling indices of tomato plants grown in soil treated with ammonium sulfate or chicken litter in combination with the neem extract or a chemical nitrification inhibitor were far lower than those of plants grown in the control soil or in soil treated with chicken litter, neem extract or nitrification inhibitor alone. However, plants grown in the microplots showed only slight reductions in galling, probably because the soil amendments were inadequately mixed compared to their application in the pot experiments. The extended exposure of nematodes to ammonia as a result of nitrification inhibition by the neem extracts appeared to be the cause of the enhanced nematicidal activity of the ammonia-releasing amendments.     

Enhancement of nematicidal potential through cloning and expression of chitinase gene from Bacillus subtilis subsp. Subtilis BTN7A strain

A gene encoding chitinase from B. subtilis has been isolated after optimization of PCR conditions. It was cloned with two different prometers, T7 promoter of the pJET1.2/blunt cloning vector and the SP6 promoter of pGEM®-T Easy vector. After transforming E. coli DH5α, two transformants were selected, CHI-NRC-4 from the first vector and T-CHI-NRC-6 from the second vector, and used for further studies. The complete CDS sequence of chitinase gene was determined and submitted to GenBank with the accession number KX268692.1. Culture supernatants of E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) were investigated for their inhibitory effect on M. javanica egg hatch under laboratory conditions. Result showed up to 96% inhibition in egg hatching due to both E. coli transformants as compared to control which reflect the same expression efficiency of both used prometers. A greenhouse experiment was carried out to evaluate the nematicidal effect of culture supernatants of the two transformts E. coli (CHI-NRC-4) and E. coli (T-CHI-NRC-6) against M. javanica infected eggplant. Obtained results showed a significant reduction in nematode population in soil and roots and enhancement in eggplant growth parameters as compared to control.