J2 Juveniles Research Articles

Bacillus, Pseudomonas and Serratia control Meloidogyne incognita (Rhabditida: Meloidogynidae) and promote the growth of tomato plants

Plant-parasitic nematodes cause significant losses in agriculture worldwide. Among these parasites, the root-knot nematode (Meloidogyne spp.) stands out. Consequently, control methods are being developed to combat this pest. Among these methods is biological control, the main agents of which are bacteria. This study aimed to evaluate the impact of 102 bacterial isolates on the mortality of Meloidogyne incognita J2 juveniles, hypothesizing that certain isolates could effectively control the nematode and promote tomato plant growth. Five experiments were conducted to test this hypothesis. First, the effect of the 102 bacterial isolates on J2 mortality was assessed under laboratory conditions. Second, five isolates (Bacillus cereus IBCBb130, Bacillus cereus IBCBb116, Pseudomonas aeruginosa IBCBb122, Bacillus proteolyticus IBCBb136, and Serratia sp. IBCBb118), which showed >68% J2 mortality, were evaluated over 72 h. Third, these isolates were tested at seven different concentrations. Fourth, their efficacy in controlling M. incognita on tomato plants was assessed in a greenhouse setting. Fifth, their potential to promote tomato plant growth without nematode inoculation was evaluated. The results revealed that the 102 bacterial isolates caused mortality ranging from 2 to 79.57% for M. incognita J2. At a 10% dilution, five isolates (B. cereus IBCBb130, P. aeruginosa IBCBb122, B. cereus IBCBb116, B. proteolyticus IBCBb136, and Serratia sp. IBCBb118) maintained mortality rates above 68%. Among these strains, B. cereus IBCBb130 and P. aeruginosa IBCBb122 were particularly effective, with B. cereus IBCBb130 showing high mortality rates under laboratory conditions and P. aeruginosa IBCBb122 significantly reducing nematode populations in greenhouse pots. Additionally, Serratia sp. IBCBb118 demonstrated notable potential for promoting tomato plant growth. In conclusion, specific bacterial isolates exhibit strong potential for the biocontrol of M. incognita and enhancement of tomato plant growth, suggesting a viable alternative to chemical nematicides. These findings provide insight into the development of sustainable agricultural practices through targeted manipulation of the soil microbiota. Future research should explore the underlying mechanisms of these interactions and their long-term effects on crop yield and soil health.

Multistress‐tolerant Pseudomonas aeruginosa SSVP3 uses multiple strategies to control Meloidogyne incognita in tomato

AbstractTomato is the most important horticultural crop, and India is its second largest producer; however, the plant‐pathogenic nematode Meloidogyne incognita is a serious pest of tomato, causing detrimental losses in its production. This study is focused on the use of multistress‐tolerant Pseudomonas aeruginosa SSVP3 to control this devastating nematode and attempts to decipher the roles of its metabolites and volatile organic compounds (VOCs) during this interaction. The supernatant of P. aeruginosa SSVP3 caused 58% mortality in M. incognita J2 juveniles. The major nematicidal metabolites, identified using liquid chromatography‐mass spectrometry, were pyocyanin, pyoluteorin, pyochelin, benthocyanin and phenazines. The VOCs secreted by P. aeruginosa SSVP3, which were identified via gas chromatography–mass spectrometry (HS‐SPME‐GC–MS) in a mixture, caused 98% mortality in M. incognita J2 juveniles. In a pot experiment, seedling root treatment with P. aeruginosa SSVP3 activated the induced systemic response in tomato plants to M. incognita by increasing the activity of defence and antioxidant enzymes as well as the proline and phenolic contents. The malondialdehyde content in P. aeruginosa SSVP3‐treated plants decreased as compared to those in the other treatment groups. The number of galls formed and the number of endoparasitic stages of M. incognita in the untreated nematode control plants were much greater (19 and 35) than those in the P. aeruginosa SSVP3‐treated (0.33 and 1) and chemically treated groups (0.67 and 1.33). These results indicate that P. aeruginosa and its secreted metabolites and VOCs have a high potential for controlling nematodes.

Potential of 2-Hydroxyacetophenone Derivatives and Simple Phenol's for the control of Meloidogyne javanica

Objective: In this study, a series of synthetic hydroxylated (5-10) and prenylated (10-17) acetophenones, chalcones (18-20) and a flavone (21) have been synthesized and evaluated on the nematode Meloidogyne javanica. Additional phenolic compounds, including salicylic acid, isopropyl salicylate, thymol, carvacrol, cumic acid, eugenol, vanillin, and vanillic acid (22-29), were also tested. Methods: Compounds 5-21 have been synthesized by a sequence of reactions and were isolated and identified by 1H-NMR, 13C-NMR, and HRMS. The in vitro nematicidal effects of the compounds were tested on J2 juveniles. The nematicidal activity was confirmed by their egg hatching inhibition effects, tested at the minimum lethal concentration (minimum dose to give 100% J2 mortality). Results: Compounds 5-18 have been previously reported and 19-21 are described here for the first time. The 2´-hydroxylated acetophenones (5-9) showed strong nematicidal effects, being 7-9 the most active. Salicylic (22) and cumic (26) acids were effective compounds. Compounds 7, 12, 22-26, active on M. javanica J2 juveniles, were also egg hatching inhibitors. Conclusions: A C-2/C-4 dihydroxylation or C-2/C-4/C-3 trihydroxylation of the acetophenone moiety improved the activity, while introducing a hydrophilic isoprene chain (10-17) eliminated the effect, with only one prenylated acetophenone (12) being active. Among the additional phenolic compounds (22-29), these with a carboxyl group instead of the acyl side were better nematicides. These results generate molecular leads for developing new nematicidal phenolic compounds.

NEMATOCIDAL PROPERTIES OF RED SILK COTTON (Bombax costatum Pellegr. & Vuil.) FLOWER AGAINST ROOT-KNOT NEMATODE, (Meloidogyne javanica (Treub. 1885) CHITWOOD 1949

A study was conducted in Jalingo Nigeria to determine the nematocidal activity of water extract of flower powder of Bombax costatum against the eggs and J2 juveniles of Meloidogyne javanica in the laboratory. For both egg hatch inhibition and juvenile mortality, 15 Petri-dishes were used for each and were arranged in Complete Randomized Design (CRD). For egg hatch inhibition test, 500 suspended in 10 ml distilled water were dispensed into each of 15 Petri-dishes used. Five treatments used included crude extract (stock solution) of B. costatum, 5 ml, 10 ml and 15 ml dilutions and control which were also dispensed into the petri dishes. Hatched nematodes were counted after three days. For juvenile mortality test, 500 J2s suspended in 10 ml distilled water were dispensed into each of 15 Petri-dishes used. The five treatments used included crude extract (stock solution) of B. costatum, 5 ml, 10 ml and 15 ml dilutions and control which were also dispensed into the petri dishes. Dead nematodes were counted after every day for three days. Data collected was analyzed. Results showed that the 100% concentration (crude extract) treatment gave the highest egg hatch inhibition and juvenile mortality of 89.33% and 94.33% respectively. This is an indication of the potential of flower powder of B. costatum to limit these inoculums of M. javanica and therefore reduce their damage potential. Pot and field trials will be needed before can be recommended for wider use.

Trichoderma erinaceum AND Trichoderma virens IN THE CONTROL OF Meloidogyne incognita IN Solanum lycopersicum

In this study, the antagonistic potential of Trichoderma erinaceum 10-15 and T. virens 32-09 against the nematode Meloidogyne incognita was evaluated in tomato (Solanum lycopersicum L.) plants. A solution of 106 conidia mL-1 of each strain was inoculated on tomato plants, and the variables of nematode control, galling index, eggs per gram of root, and females per gram of root were determined. The in vitro antagonism of Trichoderma filtrates on the hatching of eggs and J2 juveniles of M. incognita was evaluated. The results were used for analysis of variance and comparison of means using the Tukey method (p ≤ 0.05). Control variables of the phytopathogen on S. lycopersicum were determined by measuring the area under the disease progress curve (AUDPC), the Weibull parameter, the coefficient of determination, and the final severity of root damage. T. erinaceum 10-15 showed a greater inhibitory effect on M. incognita, relative to T. virens 32-09. Both strains evaluated showed significant differences in comparison to the uninoculated control, resulting in a 60 % reduction in juvenile stage mobility and egg hatching, as well as greater control of the phytonematode in the AUDPC variables, Weibull parameter, and final severity under controlled conditions. The strain T. erinaceum 10-15 showed a better antagonistic effect on egg formation at 68 days, at 60.3 %, and on reducing the number of M. incognita females with at 60 days, at 80.6 %. Both T. erinaceum 10-15 and T. virens 32-09 were found to be potential biocontrol agents of M. incognita in S. lycopersicum.

Molecular Characterization and Functional Analysis of GPCR Gene Bx-srh-1 in Pinewood Nematode (Bursaphelenchus xylophilus)

Bursaphelenchus xylophilus, also known as the pine wood nematode (PWD), which causes pine wilt disease (PWD), is one of the most devastating diseases affecting pine forests globally. G protein-coupled receptors (GPCRs) are crucial in many biological processes and serve as privileged points of communication between cells and the surrounding environment. α-pinene has been found to play a crucial role in combating the infection, colonization, and early stages of pathogenesis caused by B. xylophilus. In this study, we investigated the molecular characteristics and biological functions of the GPCR gene Bx-srh-1 in B. xylophilus. Fluorescence in situ hybridization (FISH) was performed to determine the spatial expression patterns of Bx-srh-1 in B. xylophilus. The results indicated that Bx-srh-1 is expressed in the intestine and subcutaneous tissues of J2 and J3 juveniles and in the spicules of adult males and vulvae of adult females. RNA interference (RNAi) was used to analyze Bx-srh-1 gene function, and we examined the expression patterns of Bx-srh-1 in B. xylophilus under α-pinene stress. The RNA interference indicated that Bx-srh-1 was involved in the reproductive ability and pathogenicity of B. xylophilus; the expression levels of Bx-srh-1 significantly increased after the exposure to α-pinene for 12 h, and they peaked at 48 h. Silencing Bx-srh-1 may therefore lead to a reduction in B. xylophilus reproduction and pathogenicity. These results demonstrate that Bx-srh-1 is related to the feeding behavior, reproduction, pathogenicity, and resistance to α-pinene process of B. xylophilus.

Volatile Organic Compounds of Bacillus velezensis GJ-7 against Meloidogyne hapla through Multiple Prevention and Control Modes.

The Bacillus velezensis GJ-7 strain isolated from the rhizosphere soil of Panax notoginseng showed high nematicidal activity and therefore has been considered a biological control agent that could act against the root-knot nematode Meloidogyne hapla. However, little was known about whether the GJ-7 strain could produce volatile organic compounds (VOCs) that were effective in biocontrol against M. hapla. In this study, we evaluated the nematicidal activity of VOCs produced by the fermentation of GJ-7 in three-compartment Petri dishes. The results revealed that the mortality rates of M. hapla J2s were 85% at 24 h and 97.1% at 48 h after treatment with the VOCs produced during GJ-7 fermentation. Subsequently, the VOCs produced by the GJ-7 strain were identified through solid-phase micro-extraction gas chromatography mass spectrometry (SPME-GC/MS). Six characteristic VOCs from the GJ-7 strain fermentation broth were identified, including 3-methyl-1-butanol, 3-methyl-2-pentanone, 5-methyl-2-hexanone, 2-heptanone, 2,5-dimethylpyrazine, and 6-methyl-2-heptanone. The in vitro experimental results from 24-well culture plates showed that the six volatiles had direct-contact nematicidal activity against M. hapla J2s and inhibition activity against egg hatching. In addition, 3-methyl-1-butanol and 2-heptanone showed significant fumigation effects on M. hapla J2s and eggs. Furthermore, all six of the VOCs repelled M. hapla J2 juveniles in 2% water agar Petri plates. The above data suggested that the VOCs of B. velezensis GJ-7 acted against M. hapla through multiple prevention and control modes (including direct-contact nematicidal activity, fumigant activity, and repellent activity), and therefore could be considered as potential biocontrol agents against root-knot nematodes.

First report of ryegrass cyst nematode, Heterodera mani, in Tasmania, Australia.

Cyst nematodes of the genus Heterodera are a major group of sedentary plant parasites causing a significant economic impact, restricting production and market access globally (Moens et al. 2018). The ryegrass cyst nematode Heterodera mani is in the Avenae group and is found predominantly in pastures and grasslands in Europe, California, and South Africa. It was first described by Mathews (1971) from Northern Ireland. Known hosts are grasses (family Poaceae), principally Lolium perenne (perennial ryegrass), but also Dactylis glomerata (cat grass) and Festuca pratensis (meadow fescue) (Subbotin et al. 2010). Mowat (1974) reported that H. mani causes negligible damage to the yield of L. perenne in pot trials; however, Maas & Brinkman (1982) determined that it may cause significant damage to spring and autumn-sown perennial ryegrass in field conditions. During a routine examination for potato cyst nematode from a farm near Mawbanna in north-west Tasmania, Australia, several pale to dark brown Heterodera cysts were extracted that were lemon shaped with the presence of a small vulval cone at the posterior end and a distinct neck. The J2 (n=20) stylet length ranged from 24-26 µm with round knobs deeply concave anteriorly, hyaline tail length was 37-42 µm, true tail length ranged from 59-68 µm and total body length varied from 526-559 µm. All the above characters match those described for H. mani (Subbotin et al. 2010). To verify this identification, DNA was extracted from five individual J2 juveniles from a single cyst using QIAamp DNA micro kit (Qiagen®), and two gene regions amplified: internal transcribed spacer region of ribosomal RNA (ITS-rRNA) with primer pair AB28 and TW81 and cytochrome oxidase 1 (CO1) with primer pair JB3 and JB5 (Bowles et al. 1992; Curran et al. 1994; Derycke et al. 2005). One PCR reaction contained 10 µM (1 µl each) of each primer, 12.5 µl of OneTaq® DNA Polymerase and 5 µl of DNA template with a final volume of 25 µl. PCR products were sent for purification and Sanger sequencing at Macrogen (Seoul, Rep. of Korea). All resulting sequences were trimmed, aligned, and analysed using Geneious Prime® 2022.0.1 (www.geneious.com). Five ITS sequences (accessions ON402852-ON402856) and five CO1 sequences (accessions ON402857-ON402861) were submitted to GenBank. These ITS sequences were very similar to each other and exhibited 99.16-100% similarity with that of H. mani isolate from Hamminkeln, Germany (AY148377) (Subbotin et al. 2018). The CO1 sequences exhibited 98.96-100% similarity with that of H. mani isolate from Washington, USA (MG523097) (Subbotin et al. 2003). Obtained sequences were mapped to reference sequences downloaded from NCBI GenBank and maximum likelihood phylogenetic trees were calculated. Due to the lack of further living nematode material, pot experiments were not performed. Such experiments are not feasible in Tasmania currently and transfer of live nematode material to the Australian mainland presents logistic and legal issues. However, morphological and molecular evidence for species determination of H. mani was unequivocal and contributes to the list of cyst nematode species present in Australia. This is the first detection of H. mani in Australia and is a range extension of the species from North America, Africa, and Europe to Australia. The nematode may cause damage to perennial ryegrass in Australia, however, impact on yield still needs to be investigated.

The soil mite Cunaxa capreolus (Acari: Cunaxidae) as a predator of the root-knot nematode, Meloidogyne incognita and the citrus Nematode, Tylenchulus semipenetrans: Implications for biological control

Plant-parasitic nematodes (PPNs) are dangerous pests, causing serious losses to the world’s agricultural crops. As soil-dwelling predaceous mites are known as potential biological control agents against many pests, we investigated the interactions between the cunaxid mite, Cunaxa capreolus (Berlese), and two plant parasites (root-knot nematode, Meloidogyne incognita (Kofoid and White) and the citrus Nematode, Tylenchulus semipenetrans Cobb under laboratory conditions. The predatory mite C. capreolus completed its life-span when fed on egg masses EM and second-stage juveniles J2 of M. incognita and J2 juveniles of T. semipenetrans as food sources in the laboratory in sealed arenas at 32ºC, 60% relative humidity in the dark. Males developed slightly faster than females irrespective of different prey. Adult females lived longer than males and showed a higher rate of food consumption. Life table parameters indicated that feeding C. capreolus on J2 juveniles of M. incognita and J2 juveniles of T. semipenetrans led to the highest reproduction rate (r<sub>m</sub>= 0.185 and 0.167 females/female/day), while feeding on EM of M. incognita gave the lowest reproduction rate (r<sub>m</sub>= 0.085). The results show that C. capreolus multiplied rapidly when juveniles of M. incognita and T. semipenetrans were offered as prey, indicating the mite’s potential for regulating population densities of these two pests. Future research should focus on understanding the crop and soil management applications required to enable this cunaxid mite and other predatory species to thrive. The implications of these results on biological control of plant parasitic nematodes are discussed.

Nematicidal activity of native Bacillus thuringiensis against the root knot nematode, Meloidogyne incognita (Kofoid and White)

In this study, potential of the native Bacillus thuringiensis Berliner was investigated against the root knot nematode, Meloidogyne incognita (Kofoid and White) under laboratory and greenhouse conditions. Fourteen out of 50 Bt isolates obtained from root zone of vegetable crops of north western zone of Tamil Nadu, India, were found to be encoded with nematicidal cry genes, confirmed by gene-specific PCR. Nine different Cry protein profiles were obtained from native Bt. Spore-crystal mixtures of 6 isolates out of 14 (at 0.5%) showed 100% inhibition to J2 juveniles emergence from egg masses of M. incognita within 72 h of treatment. Analysis by SEM revealed that fluffy egg masses were observed in untreated samples due to hatching, whereas egg masses were covered by gelatinous matrix, when treated with spore-crystal mixture of nematicidal Bt. Two (BC and BD) of the 6 isolates were selected based on their highest nematicidal activity against J2 of M. incognita with LC50 values of 0.12 and 0.23 μg/ml of protein. The spore-crystal mixtures of isolates BC and BD when applied to tomato plants under greenhouse conditions exhibited the enhanced biocontrol potential by suppressing number of egg masses, reduction of female population and decreased root gall index, when compared to control and chemical treatments.

Annulohypoxylon sp. FPYF3050 produces volatile organic compounds against the pine wood nematode, Bursaphelenchus xylophilus

Summary Natural volatiles released by the fungus, Annulohypoxylon sp. FPYF3050, were evaluated against the pine wood nematode (PWN), Bursaphelenchus xylophilus. Our results showed that volatile organic compounds (VOCs) caused 64.1 and 58.4% mortality of second-stage juveniles (J2) and mixed-stages (eggs, J2, third- and fourth-stage juveniles, and adults) of populations of PWN, but no inhibitive effects were detected on nematode eggs in the experiment. Analysis of the gases within the Petri plate by gas chromatography-mass spectrometry (GC-MS) showed a yield of an unique volatile with dominant 1,8-cineole in 77.4% relative area (RA) after 72 h treatment of nematodes with Annulohypoxylon sp. FPYF3050 and Botrytis cinerea. The commercial 1,8-cineole at concentrations of 2, 5, 10 and 15 μl ml−1 was applied to examine nematicidal activity. The results showed that 1,8-cineole had a 40-100% inhibition on the nematode eggs during 48 h treatment, more than 82.9% mortality of J2 after 24 h, 48 h and 96 h, and 18.7-91.9% mortality of the mixed-stage population, depending on the period after exposure. This result indicates that 1,8-cineole in the volatile gas emissions of Annulohypoxylon sp. FPYF3050 may play a crucial inhibitory effect on the pine wood nematode The nematicidal volatile gas from fungi may provide a useful biocontrol agent for controlling B. xylophilus.

Small RNA Sequencing Reveals Regulatory Roles of MicroRNAs in the Development of Meloidogyne incognita.

MicroRNAs (miRNAs) are an extensive class of small regulatory RNAs. Knowing the specific expression and functions of miRNAs during root-knot nematode (RKN) (Meloidogyne incognita) development could provide fundamental information about RKN development as well as a means to design new strategies to control RKN infection, a major problem of many important crops. Employing high throughput deep sequencing, we identified a total of 45 conserved and novel miRNAs from two developmental stages of RKN, eggs and J2 juveniles, during their infection of cotton (Gossypium hirsutum L.). Twenty-one of the miRNAs were differentially expressed between the two stages. Compared with their expression in eggs, two miRNAs were upregulated (miR252 and miRN19), whereas 19 miRNAs were downregulated in J2 juveniles. Nine miRNAs were expressed at high levels, with >1000 reads per mapped million (RPM) sequenced reads in both eggs and J2 juveniles (miR1, miR124, miR2-3p, miR252, miR279, miR57-5p, miR7904, miR87, and miR92). Three miRNAs were only expressed in eggs (miR4738, miRN3, and miRN5). These differentially expressed miRNAs may control RKN development by regulating specific protein-coding genes in pathways associated with RKN growth and development.

Biological control of the root-knot nematode Meloidogyne incognita on tomatoes and carrots by plant growth-promoting rhizobacteria

In the current study 27 plant growth-promoting rhizobacteria (PGPR) strains were screened for biocontrol activity against Meloidogyne incognita on 6-week old carrot seedlings that were inoculated with a suspension of J2 juveniles five days after treatment with the bacteria. Five of the PGPR strains, namely Bacillus firmus T11, Bacillus aryabhattai A08, Paenibacillus barcinonensis A10, Paenibacillus alvei T30, and Bacillus cereus N10w, caused 86.0, 85.2, 84.6, 81.5 and 82.1% reduction in gall numbers, respectively. In a subsequent greenhouse experiment on carrots, treatment with strain T30 caused a significant reduction in gall index and egg mass index compared to the control. The reduction in gall numbers caused by strain T30 was not statistically different from that obtained with CropGuard (furfural). Treatment with B. aryabhattai A08 gave the best results on tomato, significantly reducing gall index and egg mass index compared to the control. In vitro experiments with all five abovementioned strains resulted in second-stage juvenile (J2) paralyses. Induction of resistance was not observed in a split-root experiment conducted in the greenhouse, suggesting that secondary metabolites produced by the bacterial strains are responsible for the biocontrol activity. It is concluded that the bacterial strains P. alvei T30 and B. aryabhattai A08 have potential as biological control agents of M. incognita on carrots and tomatoes respectively.

Multiple Modes of Nematode Control by Volatiles of Pseudomonas putida 1A00316 from Antarctic Soil against Meloidogyne incognita.

Pseudomonas putida 1A00316 isolated from Antarctic soil showed nematicidal potential for biological control of Meloidogyne incognita; however, little was known about whether strain 1A00316 could produce volatile organic compounds (VOCs), and if they had potential for use in biological control against M. incognita. In this study, VOCs produced by a culture filtrate of P. putida 1A00316 were evaluated by in vitro experiments in three-compartment Petri dishes and 96-well culture plates. Our results showed that M. incognita juveniles gradually reduced their movement within 24–48 h of incubation with mortality ranging from 6.49 to 86.19%, and mostly stopped action after 72 h. Moreover, egg hatching in culture filtrates of strain 1A00316 was much reduced compared to that in sterile distilled water or culture medium. Volatiles from P. putida 1A00316 analysis carried out by solid-phase micro-extraction gas chromatography–mass spectrometry (SPME-GC/MS) included dimethyl-disulfide, 1-undecene, 2-nonanone, 2-octanone, (Z)-hexen-1-ol acetate, 2-undecanone, and 1-(ethenyloxy)-octadecane. Of these, dimethyl-disulfide, 2-nonanone, 2-octanone, (Z)-hexen-1-ol acetate, and 2-undecanone had strong nematicidal activity against M. incognita J2 larvae by direct-contact in 96-well culture plates, and only 2-undecanone acted as a fumigant. In addition, the seven VOCs inhibited egg hatching of M. incognita both by direct-contact and by fumigation. All of the seven VOCs repelled M. incognita J2 juveniles in 2% water agar Petri plates. These results show that VOCs from strain 1A00316 act on different stages in the development of M. incognita via nematicidal, fumigant, and repellent activities and have potential for development as agents with multiple modes of control of root-knot nematodes.

Mineral composition variation in the shells of freshwater mussel Anodonta woodiana at different growth stages

The levels of mineral element Na, Mg, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Mo, Al, As, Ag, Cd, and Tl were quantified in the whole shells of the freshwater bivalve Anodonta woodiana at three different growth stages (i.e. J1 juveniles of 1 month old, J2 juveniles of 3.5 months old, and adults of 36 months old). The concentrations of Na and Al were different between different growth stages (p < 0.05). The highest Na concentrations (2715 ± 86 μg/g dry weight) were found in J2 juveniles. The highest Al concentrations (303.9 ± 5.95 μg/g dry weight) were found in J1 juveniles. Manganese concentrations (517.0 ± 47.98 μg/g dry weight) were significantly higher in J2 juveniles than in J1 juveniles (432.3 ± 9.87 μg/g dry weight) (p < 0.05). Copper concentrations (27.32 ± 0.15 μg/g dry weight) were significantly higher in J1 juveniles than in J2 juveniles (26.21 ± 0.86 μg/g dry weight) and adults (24.74 ± 1.43 μg/g dry weight) (p < 0.05). Burdens of Na, Ca, Mn, Fe, Co, Cu, Mo, Ag, and Tl were positively correlated with the shell length (p < 0.05). These findings can possibly contribute to an understanding of elemental requirements for shell growth and, hence, facilitate improvement of survival and growth rates during artificial mussel culture.

Biocidal and Antinemic Properties of Aqueous Extracts of Ageratum and Coccinia Against Root-Knot Nematode, Meloidogyne Incognita In Vitro

Plants are the one of the glorious and fascinating source of natural biopesticides. The present investigation was carried out for pytochemical analysis and to assess the nematicidal and nematostatic potential of aqueous extract of some plant parts viz., Ageratum conyzoides and Coccinia grandis against hatching and nematicidal behavior of root-knot nematode, Meloidogyne incognita in vitro conditions. The second stage juveniles (J2) were exposed at 24, 48 and 72 hours in different concentration (S, S/2, S/10, and S/100) of plant extracts. During in vitro condition inhibition of egg hatchability and J2 juveniles’ mortality varied according to the concentration of plant extract. The extract of Ageratum conyzoides leaves and stem exhibited highly promising mortality (98-100%) after 72 hours of exposure periods while the plant extract of C. grandis leaves and fruit showed minimum mortality (56-60%) after 24 hours of exposure periods. There was a gradual decrease in egghatching with an increase in the concentration of aqueous extract of plants. A. conyzoides leaves and stem extract elucidating most effectiveness in reducing egghatching and increase in mortality of J2 juveniles of M. incognita. Concentration of the extract was directly proportional to the mortality of second stage juveniles and inversely proportional to the egghatching. Phytochemical analysis in various solvent of different polarity such as ethanol, methanol, acetone, chloroform, petroleum and water marked the impressive outcome of alkaloids, saponins, tannins, flavonoids, protein, amines, glycosides, carbohydrate, steroids, mucilage, gums, terpenoids and phlobatannins in the plant extracts. The aqueous extract of Ageratum conyzoides showed maximum egg inhibition and J2 juvenile mortality due to the presence of phytoconstituents such as alkaloid, tannins, phenol, saponins, glycosides, flavonoids, carbohydrate, protein, mucilage/gum and phlobatannins while Coccinia grandis showed minimum egg inhibition and juvenile mortality.

Gene expression analysis in Musa acuminata during compatible interactions with Meloidogyne incognita.

Endoparasitic root-knot nematodes (RKNs) ( Meloidogyne spp.) cause considerable losses in banana ( Musa spp.), with Meloidogyne incognita a predominant species in Cavendish sub-group bananas. This study investigates the root transcriptome in Musa acuminata genotypes 4297-06 (AA) and Cavendish Grande Naine (CAV; AAA) during early compatible interactions with M. incognita . Roots were analysed by brightfield light microscopy over a 35 d period to examine nematode penetration and morphological cell transformation. RNA samples were extracted 3, 7 and 10 days after inoculation (DAI) with nematode J2 juveniles, and cDNA libraries were sequenced using lllumina HiSeq technology. Sequences were mapped to the M. acuminata ssp. malaccensis var. Pahang genome sequence, differentially expressed genes (DEGs) identified and transcript representation determined by gene set enrichment and pathway mapping. Microscopic analysis revealed a life cycle of M. incognita completing in 24 d in CAV and 27 d in 4279-06. Comparable numbers of DEGs were up- and downregulated in each genotype, with potential involvement of many in early host defence responses involving reactive oxygen species and jasmonate/ethylene signalling. DEGs revealed concomitant auxin metabolism and cell wall modification processes likely to be involved in giant cell formation. Notable transcripts related to host defence included those coding for leucine-rich repeat receptor-like serine/threonine-protein kinases, peroxidases, thaumatin-like pathogenesis-related proteins, and DREB, ERF, MYB, NAC and WRKY transcription factors. Transcripts related to giant cell development included indole acetic acid-amido synthetase GH3.8 genes, involved in auxin metabolism, as well as genes encoding expansins and hydrolases, involved in cell wall modification. Expression analysis in M. acuminata during compatible interactions with RKNs provides insights into genes modulated during infection and giant cell formation. Increased understanding of both defence responses to limit parasitism during compatible interactions and effector-targeted host genes in this complex interaction will facilitate the development of genetic improvement measures for RKNs.

Evaluation of Some Botanicals and Trichoderma harzianum for the Management of Tomato Root-knot Nematode (Meloidogyne incognita (Kofoid and White) Chit Wood)

Root-knot nematode disease caused by Meloidogyne incognita (Kofoid and White) Chit wood) is one of the major constraints for successful cultivation of tomato (Lycopersicon esculentum Mill.) in Ethiopia. Hence, the present study was conducted to evaluate the effect of leaf and seed extracts of four botanicals viz., Rape seed (Brassica napus L.), Lantana (Lantana camara L.), African marigold (Tagetes erecta L.) and Neem (Azadirachta indica L.) at 5% and 10% concentrations and T. harzianum at 5% plus control were tested on root-knot nematode under in vitro and also to evaluate their against root-knot nematode development and their role on plant growth parameters of tomato under in vivo condition. Plant extracts were more effective and significantly inhibited egg hatching and immobilizing the J2 larval mortality of M. incognita than T. harzianum. Aqueous extract of all the tested plants inhibited egg hatching of nematode and resulted 84.67-100% mortality of the J2 juveniles of M. incognita in vitro at the 10% concentration after 72 h of exposure time. There were no significant differences among the treatments of rape seed leaf (84.7%) at 10% concentration and Lantana camara (87%), African marigold (86.3%) and Neem leaf (85%) at 5% concentration after 72 h. Aqueous seed extracts of A. indica more significantly inhibited egg hatching and larva mortality of the J2 of M. incognita in vitro at the 10% concentration and immobilized by 89, 93 and 100% after 24, 48 and 72 h of exposures, respectively, while at similar concentration of T. erecta, B. napus and L. camara leaf extracts exhibited 92, 89 and 93.2% inhibition of egg hatching and 75, 62.1 and 73% larval mortality, respectively. The effect of different botanicals and T. harzianum singly and in combination were studied for the management of tomato root-knot nematode under greenhouse condition. There was a significant difference in the reduction of root-knot nematode incidence, root-knot nematode population, nematode reproduction rate (NRR), number of galls and egg masses per plant were recorded. In pot culture condition, the application of leaf extract of individual plant in the presence of the nematode significantly enhanced the growth of tomato seedlings in comparison to the control. A significant increase in plant height, shoot weight and root weight of the seedlings were observed at the 10% concentration of leaf extracts in comparison to control. There was a significant difference in the reduction of root-knot nematode population, nematode reduction rate, number of galls and egg masses per plant of L. camara combined with T. harzianum. The mean fruit weight and total yield were observed highest in the combination treatment of L. camara combined with T. harzianum. This study results revealed that the test plants are readily available to farmers at no cost and able to reduce nematode population below economic threshold.

English

Large number of second-stage juveniles of root-knot nematode, Meloidogyne incognita was prepared for scanning electron microscopy over a millipore filter (pore size 0.45 &micro;m). The J2 juveniles were quickly immobilized and killed before fixation using the cryo-fixation technique followed by the primary fixation in 2.5% glutaraldehyde pre-cooled at 4-8&deg;C and then secondary vapor fixation in 1% osmium tetraoxide and drying with the series of ethanol without subsequent critical point drying. The specimens were vacuum dried in a desiccator for a period of 24-48 h. Cryo-fixation emphasizes the need to quickly stop cellular activity and thus prevent any surface artifacts that occur due to conventional fixation techniques. &nbsp; Key words: Scanning electron microscopy, cryo-fixation, root-knot nematode, Meloidogyne incognita, surface morphology.

Wystąpienie guzaka północnego Meloidogyne hapla Chitwood, 1949 (Nematoda: Meloidogynidae) w sadzeniakach na terytorium Polski

During official controls of seed potatoes performed by the State Plant Health and Seed Inspection Service in 2011–2012, the presence of the northern root-knot nematode (Meloidogyne hapla Chitwood, 1949) was revealed in 21 tuber samples originating from the area of Lubelskie Voivodeship in Poland. In 2011 the level of infestation was 2.61% (11 from among 422 tested samples were infested) and in 2012 – 2.81% (the nematodes were found in 10 samples from among 356 tested). The results of the morphological and morphometric analyses of females and J2 juveniles isolated from all 21 positive samples were confirmed by results of conducted molecular tests (PCR-ITS – Polymerase Chain Reaction-Internal Transcribed Spacer, PCR-SCAR – Polymerase Chain Reaction-Sequence Characterized Amplified Regions and PCR-RFLP – Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) which simultaneously excluded presence of quarantine root-knot nematode species Meloidogyne chitwoodi (Golden et al. 1980) and M. fallax (Karssen 1996). Podczas urzędowych kontroli sadzeniaków ziemniaka prowadzonych przez Państwową Inspekcję Ochrony Roślin i Nasiennictwa w latach 2011–2012 stwierdzono obecność guzaka północnego (Meloidogyne hapla Chitwood, 1949) w 21 próbach bulw pochodzących z terenu województwa lubelskiego. W roku 2011 stopień porażenia wynosił 2,61% (porażonych było 11 spośród 422 badanych prób), natomiast w 2012 r. – 2,81% (nicienie stwierdzono w 10 próbach spośród 356 badanych). Wyniki analiz morfologicznych i morfometrycznych samic i osobników młodocianych J2 nicieni wyizolowanych ze wszystkich 21 prób pozytywnych zostały potwierdzone przez wyniki przeprowadzonych testów molekularnych (PCR-ITS – Polymerase Chain Reaction-Internal Transcribed Spacer, PCR-SCAR – Polymerase Chain Reaction-Sequence Characterized Amplified Regions and PCR-RFLP – Polymerase Chain Reaction-Restriction Fragment Length Polymorphism), które jednocześnie wykluczyły przynależność nicieni do kwarantannowych gatunków Meloidogyne chitwoodi (Golden i wsp. 1980) i M. fallax (Karssen 1996).