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.     

Molecular cloning and virus-induced gene silencing of MiASB in the southern root-knot nematode, Meloidogyne incognita

Root-knot nematodes (Meloidogyne spp.), cause serious damage to agricultural production worldwide. In this study, we designed special primers based on the predicted Mitochondrial ATP synthase b subunit gene (ASB) sequence to clone the same gene in M. incognita (MiASB). The identity between the cloned MiASB and the predicted MiASB was as high as 100 %. Using the tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) system, we delivered MiASB RNAi triggers to the M. incognita feeding site on tomato seedlings, resulting in significantly fewer galls on the seedlings. Sixty days after inoculation with M. incognita, the number of root galls induced on the MiASB silence-treated seedlings was reduced by 64.3 % compared to that on the control seedlings, and reduced by 64.1 % compared to that on untreated control seedlings. This study revealed the MiASB silencing had a positive effect on the control of root-knot nematodes, and MiASB may be associated with the formation of galls caused by the nematode.     

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).     

Effects of sanitation processes on survival of Synchytrium endobioticum and Globodera rostochiensis

The two quarantine pests Synchytrium endobioticum, the causal agent of potato wart disease and Globodera rostochiensis, the yellow potato cyst nematode are currently present in Germany. Winter sporangia of Synchytrium endobioticum and cysts of Globodera rostochiensis can be spread with waste from potato processing industries, if infected tubers are processed. The German Biowaste Ordinance prescribes sanitation of organic waste before it can be used on arable land as fertilizer or filling material. Sanitation parameters prescribed by the German Biowaste Ordinance include composting for 7 days at 65°C or 14 days at 55°C or pasteurisation for 60 min at 70°C. The effect of composting and pasteurisation processes on winter sporangia of Synchytrium endobioticum and cysts of Globodera rostochiensis was tested with varying time-temperature relations. Cysts of Globodera rostochiensis were killed by composting for 7 days at 50–55°C and by pasteurisation for 30 min at 70°C. In contrast, viable winter sporangia of Synchytrium endobioticum could be extracted from sample material after composting for 70 days at 30–45°C, composting for 21 days at 50–55°C and after composting for 12 days at 60–65°C. Likewise viable winter sporangia could be extracted after pasteurisation for 90 min at 70°C and heating in a water bath at 80°C and in a dry oven at 90°C for 8 h. The parameters prescribed in the German Biowaste Ordinance are sufficient to kill cysts of Globodera rostochiensis but not sufficient to kill winter sporangia of Synchytrium endobioticum in organic waste.     

Species-specific DNA markers for identification of two root-knot nematodes of coffee: Meloidogyne arabicida and M. izalcoensis

Seven root-knot nematodes (RKN), including Meloidogyne exigua, M. incognita, M. paranaensis, M. enterolobii, M. arabicida, M. izalcoensis and M. arenaria are major pathogens of coffee crop in the Americas. Species-specific primers for their identification have been developed for five of them and constitute a fast and reliable method of identification. Here we report a PCR-based assay for specific detection of M. arabicida and M. izalcoensis. Random Amplified Polymorphic DNA fragments specific for these two species were converted into sequence characterized amplified region (SCAR) markers. PCR amplification using the SCAR primers produced a specific fragment of 300 bp and 670 bp for M. arabicida and M. izalcoensis, respectively, which were absent in other coffee-associated Meloidogyne spp. tested. SCAR primers also allowed successful amplification of DNA from single second-stage juveniles (J2), males and females. In addition, these primers were able to unambiguously detect the target species in nematode suspensions extracted from soil and roots samples, in different isolates of the same species or when used in multiplex PCR reactions containing mixtures of species. These results demonstrated the effectiveness of these SCAR markers and their multiplex use with those previously developed for M. exigua, M. incognita, M. paranaensis, M. enterolobii and M. arenaria constitute an essential detection tool. This diagnostic kit will contribute for specific J2 identification of the major RKN infecting coffee from field samples in the Americas.     

Detection of Meloidogyne incognita and Pochonia chlamydosporia by fluorogenic molecular probes*

Fluorescent molecular probes were applied for detection of the plant parasitic nematode Meloidogyne incognita and the nematode‐egg parasitic fungus Pochonia chlamydosporia var. chlamydosporia. A region in the M. incognita rDNA including ITS2 was selected for amplification and recognition with a real‐time PCR assay, based on a combination of three specific motifs, each recognized by a specific fluorescent probe. Similarly, a Scorpion probe was designed for the RT‐PCR quantification of P. c. chlamydosporia. For this purpose, the ITS‐2 rDNA gene of the fungus was sequenced from a number of Italian isolates. A conserved region unique for P. c. chlamydosporia found in the ITS‐2 rDNA gene was used. The probes allowed recognition of single juveniles of M. incognita and of the mycelium‐ or soil‐extracted fungal DNA. The potentialities of the detection procedures are discussed.     

Nematicidal activity of Ochradenus baccatus against the root‐knot nematode Meloidogyne javanica

Ochradenus baccatus is a widely distributed shrub in desert regions of the Middle East and North Africa. This plant's nematicidal activity against the root‐knot nematode Meloidogyne javanica was evaluated because it has been found to contain exceptionally high levels of glucosinolates. In in vitro assays with aqueous extracts of the plant, 100% of second‐stage juveniles were immobilized after exposure to 4% root‐core extract for 48 h; 8% root‐core extract suppressed their hatching by 87%, whereas stem, flower and root bark showed lower activity. Incorporation of root core or bark into the soil, as fresh or dry powder at 1 and 0·5% (w/w), respectively, reduced the number of nematodes recovered from the soil by 95–100%, whereas the flower and stem were much less effective. Results from further pot experiments indicated that only the root bark consistently contains nematicidal compounds which are effective in soil, whereas the nematicidal activity of the root core in soil was inconsistent. The presence of non‐volatile lipophilic and lipophobic nematicidal compounds in the root bark was suggested by extraction with different polar solvents, but these compounds do not seem to be isothiocyanates – glucosinolate‐hydrolysed compounds with nematicidal activity. Very poor host status of Ochradenus baccatus to M. javanica, M. incognita and M. hapla, but with root‐penetration rates of juveniles similar to those in tomato roots, suggest that this plant may be used as a cover plant or trap plant to reduce nematode populations in the soil.     

Thermal requirements and population dynamics of root‐knot nematodes on cucumber and yield losses under protected cultivation

Several studies were carried out to determine (i) thermal requirements for development, egg production and emergence of juveniles, and completion of the life cycle of Meloidogyne incognita and Meloidogyne javanica on cucumber, (ii) the maximum multiplication rate and the equilibrium density of root‐knot nematodes on cucumber and yield losses in pot and plastic greenhouse experiments, and (iii) the relationships between relative leaf chlorophyll content (RLCC) and relative cucumber dry top weight biomass (RDTWB) in relation to increasing nematode densities at planting (P_i) in pot experiments. Thermal requirements of M. incognita and M. javanica on cucumber did not differ, irrespective of the biological stage. In the pot experiments, M. javanica completed one generation. The maximum multiplication rate (a) was 833, and the equilibrium density (E) varied according to the effective inoculum densities. The relationship between RDTWB and P_i fitted the Seinhorst damage function model. The RLCC value at 40 or 50 days post‐inoculation also fitted the damage model and was related to RDTWB. In greenhouse experiments, conducted from 2009 to 2012, M. incognita completed three generations. The values for a and E were 1147 and 625 second stage juveniles (J2) per 250 cm³ soil, respectively. The tolerance limit was below zero, and the minimum relative yield ranged from 0·12 to 0·34.     

Nematicidal potential of materials from <Emphasis Type="Italic">Medicago</Emphasis> spp.

The nematicidal effect of soil amendments with dry top and root material from Medicago sativa and/or Medicago arborea was evaluated on the root-knot nematode Meloidogyne incognita and on the cyst nematode Globodera rostochiensis in potting mixes. All amendments suppressed root and soil population densities of both nematode species compared to non-treated and chemical controls. The suppressiveness of M. sativa differed between top and root material and among the amendment rates. In field conditions soil amendments with 20 or 40 t ha⁻¹ of a pelleted M. sativa meal increased tomato crop yield and reduced soil population densities and root galling by M. incognita. It is suggested that saponins were at least partly responsible for the nematicidal activity.     

Host suitability of Vitis rootstocks to root‐knot nematodes (Meloidogyne spp.) and the dagger nematode Xiphinema index,and plant damage caused by infections

The host suitability of commercial Vitis rootstocks commonly used in Spain (161‐49C, 41B, 1103P, 110R, 140Ru and SO4) to root‐knot nematodes (Meloidogyne arenaria, M. incognita, M. javanica) and Xiphinema index, and damage caused by nematode infection were determined under controlled conditions. The three root‐knot nematodes reproduced with a rate higher than one in all rootstocks, indicating that they are suitable hosts for these nematodes. Growth of rootstocks infected with the root‐knot nematodes was less vigorous than that of nematode‐uninfected controls in the majority of the rootstocks studied. Root infection resulted in moderate to severe root galling in all rootstocks. The shoot and main stem diameters appeared to be the most sensitive variables of damage caused by infection by Meloidogyne spp., with reduction rates from 36% and 53% in 161‐49C to 57% and 66% in 140Ru, respectively. The shoot height was not significantly affected by the root‐knot nematodes and the root fresh weight generally increased as a consequence of intensive galling. The nematode X. index caused significant root damage with a reproduction factor higher than one in all rootstocks. However, reproduction factor was significantly influenced by the rootstock and significantly decreased by about 12‐fold (5·7 to 18·1‐fold) with the increase in inoculum density from 100 to 1000 nematodes per plant. The root dry weight was reduced by X. index infections, and was the plant growth variable most affected by the nematode infection in all rootstocks at both inoculum densities. Meloidogyne arenaria, M. incognita, M. javanica and X. index, prevalent in many world vineyards, are all shown to have a damaging effect on the six tested rootstocks.     

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.     

Effect of plant elicitors on the reproduction of the root-knot nematode Meloidogyne chitwoodi on susceptible hosts

Sustainable strategies for plant-parasitic nematode control are required to reduce dependence on chemical nematicides. Foliar application of various compounds can induce a systemic defence response that reduces nematode infestation. The effects of benzothiadiazole (BTH), β-aminobutyric acid (BABA), jasmonates (cis-jasmone and methyl jasmonate) and salicylic acid (SA) in the development and reproduction of the root-knot nematode Meloidogyne chitwoodi in tomato plants were assessed. The effects of BTH and of the jasmonates were further tested on potato plants. Pot assays were conducted using tomato plants cv. Tiny Tim or potato cv. Désirée treated with foliar sprays and inoculated with 300 second stage juveniles. Nematode development and reproduction were assessed 21 and 45 days after inoculation. Treatment with SA had a negative effect on nematode development in tomato plants but did not affect reproduction and methyl jasmonate treatment was the most effective in reducing nematode penetration (58 %). Nematode development was significantly affected in potato plants sprayed with cis-jasmone. Nematode penetration was reduced by 90, 67 and 81 % in plants treated with BTH, cis-jasmone and methyl jasmonate respectively, although the reproduction factor (Rf) was only significantly lower in the BTH treatment (Rf?=?7.6) when compared to the control (Rf?=?18.1). Our results suggest that both the SA and JA pathways play an important role in plant defence mechanisms against root-knot nematode development and reproduction for both plants, and should be considered in the design of integrated pest management approaches.     

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.     

Effect of humic acid on Meloidogyne incognita (Kofoid & White) Chitwood infecting banana (Musa spp.)

Humic acid, a natural by-product from lignite coals, was evaluated for its potential to control root-knot nematode, Meloidogyne incogntia infecting banana. Hatching of M. incognita eggs was inhibited 50%–100% following incubation in 0.08%-2.0% humic acid in vitro. Exposure of juveniles (J2) of M. incognita to different humic acid concentrations significantly affected the mobility of J2 in vitro. The percent immobility of J2 increased with concentrations of humic acid up to 0.08%. In pot experiments, soil treatment with humic acid reduced the root galling in banana plantlets. All the humic acid tested concentrations (0.04%, 0.08%, 0.2% and 0.4%) significantly reduced the nematode soil density by 53.5%–56.7%, root infection by 61.9%–63.8%, egg population by 61.9%–63.8% and reproduction rate by 55.7%–56.6%. A significant improvement of growth of banana plantlets in terms of number of leaves, pseudostem height, girth, weight, root length and weight was noticed in humic acid treated pots. Humic acid did not suppress growth of the biocontrol agents Pseudomonas fluorescens and Trichoderma viride in vitro. Results suggest that soil treatment with humic acid at 0.04% not only offers significant nematode control but also improves growth of banana.     

Metabolic profiling in the roots of coffee plants exposed to the coffee root-knot nematode, Meloidogyne exigua

To understand the effect of nematode Meloidogyne exigua infestation on coffee plants, resistant and susceptible coffee seedlings were inoculated with second-stage juveniles of M. exigua, and root metabolites were studied for four time intervals at 0, 24, 48 and 96 h. During this important period for parasite establishment, the concentrations of phenols, carbohydrates, amino acids and alkaloids in the roots were measured, and hydrogen nuclear magnetic resonance spectra of the root extracts were used to identify and quantify other metabolites. One of the most striking changes was the concentration of fumaric acid on resistant plants, which varied from 59 μg g(of root)^?1 to 138 μg g(of root)^?1 during the first 24 h of the nematode inoculation. The same level of variation was observed much later (96 h) in susceptible plants. Similarly, formic and quinic acid concentrations increased more rapidly in the resistant plants compared to the susceptibles. Sucrose concentrations increased to 370 % in the first 48 h in the resistant plants but showed no significant variation in the susceptible plant. Besides, the concentration of alkaloids was much higher at 24 and 48 h in the susceptibles compared to the resistant plants. These results suggest that the higher production of sucrose as well as formic, fumaric and quinic acids, and the lower production of alkaloids by the resistant cultivar in the first 48 h after the nematode inoculation are associated with the resistance of coffee plants to M. exigua.     

Interactions Between the Potato Cyst Nematode Globodera rostochiensis and Diseases Caused by Rhizoctonia solani AG3 in Potatoes Under Field Conditions

Findings from 2 years of field experiments investigating the relationship between Globodera rostochiensis and Rhizoctonia solani on unique field sites are reported. In 2000, a field experiment was positioned on land that had previously been used for experimental work investigating integrated potato cyst nematode (PCN) management methods. This study had produced an ‘untypical’ mosaic of PCN population densities ranging from 5 to 221 eggs g⁻¹ soil. In 2001, the field experiment was conducted on a different field site and overlaid on a focus of G. rostochiensis population densities ranging from 11 to 108 eggs g⁻¹ soil. In each experiment, potatoes (cv. Désirée) were grown in plots with similar population densities of G. rostochiensis that were either uninoculated or inoculated with R. solani. A series of potato plant harvests were undertaken to investigate the effects of nematode infestation on the incidence and severity of R. solani diseases and the associated development of plants. In both experiments, a clear relationship was found between the density of G. rostochiensis juveniles present in potato roots and the incidence of stolons infected by R. solani, 6 weeks after planting. For the first time this interaction has been determined under field conditions. The results of the study suggest that the interaction between nematode and fungus is indirect and possible mechanisms are discussed.     

PCR zum halbquantitativen Nachweis von Rübenzystennematoden

Increased crop yield of nematode resistant sugar beet varieties compared to non-resistant varieties is dependent upon density of the sugar beet nematode, Heterodera schachtii, being below a certain threshold level. Therefore there is a requirement for an inexpensive quantitative laboratory test for H. schachtii. Currently the standard method to determine nematode densities relies on extraction of cysts from soil samples and their subsequent enumeration which is time-consuming. The method described in this article involves the extraction of nematodes obtained by a technique inducing the hatching of juveniles. Hatching of larvae is induced by incubation of the soil sample with “Acetox” followed by a Baermann funnel extraction. Instead of a visual estimation of nematode densities using a microscope, H. schachtii larvae are identified by a molecular diagnostic. The aim of this study is to distinguish between the three threshold infestation levels by comparing the intensities of PCR products derived from samples containing known numbers of target H. schachtii.