Marigold,Castor Bean,and Chrysanthemum as Controls of Meloidogyne incognita and Pratylenchus alleni

Root and soil populations of Meloidogyne incognita were significantly fewer from marigold, castor bean, and chrysanthemum than from tomato roots and soil, but not from fallow soil. Root populations of Pratvlenchus alleni were significantly fewer from marigold, castor bean, and chrysanthemum than from tomato: marigold had the fewest. Root populations of M. incognita and P. alleni from tomato simultaneously cultivated with marigold, castor bean, and chrysanthemum were significantly fewer than from tomato cultivated alone. Aborted giant cells and dead M. incognita (larvae and females) were observed in roots of marigold and castor bean, but not in chrysanthemum or tomato. Significantly more males than females occurred in castor bean roots. lnfcction sites of P. alleni appeared normal in all hosts. Thin-layer and column chromatography of alcoholic extracts from castor bean revealed no nematicidal thiophenc derivatives.     

Reproduction of Meloidogyne javanica on Plant Roots Genetically Transformed by Agrobacterium rhizogenes

Reproduction of Meloidogyne javanica was compared on several Agrobacterium rhizogenes-transformed root cultures under monoxenic conditions. M. javanica reproduced on all transformed roots tested; however, more females and eggs were obtained on potato and South Australian Early Dwarf Red tomato than on bindweed, Tropic tomato, lima bean, or carrot. Roots that grew at moderate rates into the agar and produced many secondary roots supported the highest reproduction. Numbers of females produced in cultures of transformed potato roots increased with increasing nematode inoculum levels, whether inoculum was dispersed eggs or juveniles. Females appeared smaller, produced fewer eggs, and were found in coalesced galls at the higher inoculum levels. The ratio between the final and initial population decreased sharply as the juvenile inoculum increased. The second-stage juvenile was preferred to dispersed eggs or egg masses for inoculation of tissue culture systems because quantity and viability of inoculum were easily assessed. Meloidogyne javanica reared on transformed root cultures were able to complete their life cycles on new transformed root cultures or greenhouse tomato plants.     

Production and Partial Characterization of Stylet Exudate from Adult Females of Meloidogyne incognita

Adult females of Meloidogyne incognita were excised from tomato roots and incubated in 0.04 M phosphate buffered saline, pH 7.4 for 18-72 hours to allow accumulation of stylet exudate. Twenty-four percent of the females produced exudate during the initial 18-hour incubation period; 70% of those females producing exudate initially produced additional exudate during the subsequent 54-hour incubation period. Analysis of exudate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least nine major protein bands. Differential staining with silver and Coomassie Brilliant Blue G-250 stains indicated that three of the bands were glycoproteins. Upon acid hydrolysis, 14 amino acids were detected in the stylet exudate. The basic amino acids lysine, histidine, and arginine comprised 21.8% of the total amino acids detected. No peroxidase activity was detected in the stylet exudates. Data presented extend and generally confirm prior work on the chemical composition of stylet exudate.     

Morphological and Molecular Characterization of Meloidogyne mayaguensis Isolates from Florida

The discovery of Meloidogyne mayaguensis is confirmed in Florida; this is the first report for the continental United States. Meloidogyne mayaguensis is a virulent species that can reproduce on host cultivars bred for nematode resistance. The perineal patterns of M. mayaguensis isolates from Florida show morphological variability and often are similar to M. incognita. Useful morphological characters for the separation of M. mayaguensis from M. incognita from Florida are the male stylet length values (smaller for M. mayaguensis than M. incognita) and J2 tail length values (greater for M. mayaguensis than M. incognita). Meloidogyne mayaguensis values for these characters overlap with those of M. arenaria and M. javanica from Florida. Enzyme analyses of Florida M. mayaguensis isolates show two major bands (VS1-S1 phenotype) of esterase activity, and one strong malate dehydrogenase band (Rm 1.4) plus two additional weak bands that migrated close together. Their detection requires larger amounts of homogenates from several females. Amplification of two separate regions of mitochondrial DNA resulted in products of a unique size. PCR primers embedded in the COII and 16S genes produced a product size of 705 bp, and amplification of the 63-bp repeat region resulted in a single product of 322 bp. Nucleotide sequence comparison of these mitochondrial products together with sequence from 18S rDNA and ITS1 from the nuclear genome were nearly identical with the corresponding regions from a M. mayaguensis isolate from Mayaguez, Puerto Rico, the type locality of the species. Meloidogyne mayaguensis reproduced on cotton, pepper, tobacco, and watermelon but not on peanut. Preliminary results indicate the M. mayaguensis isolates from Florida can reproduce on tomato containing the Mi gene. Molecular techniques for the identification of M. mayaguensis will be particularly useful in cases of M. mayaguensis populations mixed with M. arenaria, M. incognita, and M. javanica, which are the most economically important root-knot nematode species in Florida, and especially when low (<25) numbers of specimens of these species are recovered from the soil.     

Influence of Concomitant Pratylenchus brachyurus and Meloidogyne spp. on Root Penetration and Population Dynamics

Populations of Pratylenchus brachyurus on cotton were increased significantly in the presence of either Meloidogyne incognita or M. arenaria.This occurred with either simultaneous inoculation or prior invasion by M. incognita. P. brachyurus penetrated cotton roots previously invaded by, or simultaneously inoculated with, M. incognita, as well as, or better than, in the absence of M. incognita. Prior invasion by M. incognita, however, suppressed P. brachyurus populations on tomato, while it had no effect on alfalfa and tobacco. Populations of M. incognita on cotton were generally inhibited by the presence of P. brachyurus. Simultaneous inoculation with, or previous invasion by, P. brachyurus also inhibited root penetration by M. incognita. These findings emphasize the importance of host susceptibility in the study of concomitant nematode populations.     

Antioxidant Enzymes in Phytoparasitic Nematodes

Presence of different antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and ascorbate, p-phenilendiamine-pyrocathecol (PPD-PC), o-dianisidine, and guaiacol isoperoxidases, was shown in the phytoparasific nematode species Meloidogyne incognita, M. hapla, Globodera rostochiensis, G. pallida, Heterodera schachtii, H. carotae, and Xiphinema index. The activity of the enzymes tested differed among the life stages examined. SOD was present in cysts but was not detected in Meloidogyne egg masses. Catalase activity of Meloidogyne females was higher than that of preparasitic stages and cyst-nematode females. For the first time, ascorbate peroxidase was found to occur commonly in phytoparasitic nematodes, with the highest activity in the invading life-stages. In all the life stages examined, the antioxidant enzyme activities of M. hapla were markedly higher than those of M. incognita. Glutathione peroxidase was not found in the species examined.     

Nicotine Content of Tobacco Roots and Toxicity to Meloidogyne incognita

The motility of Meloidogyne incognita second-stage juveniles (J2) and their ability to induce root galls in tomato were progressively decreased upon exposure to nicotine at concentrations of 1-100 μg/ml. EC₅₀ values ranged from 14.5 to 22.3 μg/ml, but J2 motility and root-gall induction were not eliminated at 100 μg/ml nicotine. Nicotine in both resistant NC 89 and susceptible NC 2326 tobacco roots was increased significantly 4 days after exposure to M. incognita. The increase was greater in resistant than in susceptible tobacco. Root nicotine concentrations were estimated to be 661.1-979.1 μg/g fresh weight. More M. incognita were detected in roots of susceptible than in roots of resistant tobacco. Numbers of nematodes within resistant roots decreased as duration of exposure to M. incognita was increased from 4 to 16 days. Concentrations of nicotine were apparently sufficient to affect M. incognita in both susceptible and resistant tobacco roots. Localization of nicotine at infection sites must be determined to ascertain its association with resistance.     

Host plants influence susceptibility of whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) to the entomopathogenic fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae)

We quantified the tritrophic effect of host plant on the susceptibility of the sweetpotato whitefly Bemisia tabaci (Genn.) to a fungal pathogen in the laboratory. Second-instar whiteflies reared on cucumber, eggplant, tomato and bean plants for six generations were exposed to conidial suspensions of Isaria fumosorosea isolate IF-1106. Our results did not detect differences in response (proportional survival or median lethal time, LT₅₀ days) among insect populations derived from different plants that were treated with 10⁷ conidia/ml. However, at concentrations ≤ 5×10⁶ conidia/ml, whiteflies reared on bean and tomato died significantly more quickly (i.e. LT₅₀ of 4–5 days) compared with cucumber and eggplant reared populations (5–7 days). Bean and tomato-reared populations were also more susceptible to mycosis (LC₅₀ ≈ 6 × 10⁵ conidia/ml) compared with those reared on cucumber (1.9 × 10⁶ conidia/ml) and eggplant (1.5 × 10⁶ conidia/ml). A separate study confirmed that this differential response of whitefly populations to I. fumosorosea was not explained by differences in deposition rate of conidia on leaf surfaces (i.e. a dosage effect). Our findings show that host plants affect the pathogenicity and virulence of a herbivore pathogen, but depend on the rate of exposure (inoculum) applied.     

Toxicity of Leaf and Stem Extracts to Tylenchorhynchus dubius

Plant extracts, made by grinding 2 g of fresh tissue in 5 ml of water, were toxic to Tylenchorhynchus dubius and Hoplolaimus spp. Such extracts from leaves and stems of bean (Phaseolus vulgaris L.) and leaves of tobacco (Nicotiana tabacum L.) were most toxic; those from leaves of corn (Zea mays L.), tomato (Lycopersicon esculentum Mill.) and rhododendron (Rhododendron catawbiense L.) were less toxic; and extracts of bean roots were nontoxic. Nematode movement slowed markedly within 1 hr in tobacco leaf extract, and within 4 hr in bean leaf extract; both extracts completely inactivated or killed 95% of the nematodes in 24 hr. Heating leaf extract 10 min at 80 C eliminated toxicity. Absorption of fusicoccin, a phytotoxin produced by Fusicoccum amygdali Del., increased the toxicity of tomato leaf extracts, whereas water extracts of acetone-extracted powder preparations of leaves were about 15-fold more toxic than water extracts of fresh tissue. Addition of homogenized leaves of bean, tobacco and tomato to soil significantly reduced nematode populations within 3 days.     

Differential Response of Thor Alfalfa to Meloidogyne chitwoodi Races and M. hapla

Second-stage juveniles (J2) of races 1 and 2 of Meloidogyne chiiwoodi and M. hapla readily penetrated roots of Thor alfalfa and Columbian tomato seedlings; however, few individuals of M. chitwoodi race 1 were able to establish feeding sites and mature on alfalfa. Histopathological studies indicate that J2 of race 1 either failed to initiate feeding sites or they caused cell enlargement without typical cell wall thickening. The protoplasm of these cells coagulated, and juveniles of race 1 did not develop beyond the swollen J2 stage. A few females of race 1 fed on small giant cells and deposited a few eggs at least 20 and 30 days later than M. chitwoodi race 2 and M. hapla, respectively. Failure of race 1 to establish feeding sites was related to egression of J2 from the roots. The M. chitwoodi race 1 J2 egression from alfalfa roots was higher than egression of race 2 and M. hapla. Egression of J2 of M. chitwoodi races 1 and 2 from tomato roots was similar and higher than that of M. hapla. Thus egression plays an important role in the host-parasite relationship of M. chitwoodi and alfalfa.     

Comparative Studies on Root Invasion,Root Galling,and Fecundity of Three Meloidogyne spp. on a Susceptible Tobacco Cultivar

Root invasion, root galling, and fecundity of Meloidogyne javanica, M. arenaria, and M. incognita on tobacco was compared in greenhouse and controlled environment experiments. Significantly more M. javanica than M. arenaria or M. incognita larvae were found in tobacco roots at 2, 4, and 6 d after inoculation. Eight days after inoculation there were significantly more M. arenaria and M. javanica than M. incognita larvae. Ten days after inoculation no significant differences were found among the three Meloidogyne species inside the roots. Galls induced by a single larva or several larvae of M. javanica were significantly larger than galls induced by M. incognita: M. arenaria galls were intermediate in size. Only slight differences in numbers of egg masses or numbers of eggs produced by the three Meloidogyne species were observed up to 35 d after inoculation.     

Bacillus halotolerans strain LYSX1-induced systemic resistance against the root-knot nematode Meloidogyne javanica in tomato

Purpose

Determination of the nematicidal potential and mode of action of bacteria isolated from tobacco rhizosphere soil against the root-knot nematode Meloidogyne javanica in tomato plants.

Methods

Antagonistic bacteria were isolated from rhizosphere soil of tobacco infested with root-knot nematodes. Culture filtrate was used to examine nematicidal activity and ovicidal action of bacterial strains. Biocontrol of M. javanica and growth of treated tomato plants were assessed in pot experiments. To clarify whether secondary metabolites of bacteria in tomato roots induced systemic resistance to M. javanica, bacterial culture supernatants and second-stage juvenile nematodes were applied to spatially separated tomato roots using a split-root system. Bacterial strains were identified by 16S rDNA and gyrB gene sequencing and phylogenetic analysis.

Results

Of the 15 bacterial strains isolated, four (LYSX1, LYSX2, LYSX3, and LYSX4) demonstrated nematicidal activity against second-stage juveniles of M. javanica, and strain LYSX1 showed the greatest antagonistic activity; there was dose-dependent variability in nematicidal activity and inhibition of egg mass hatching by strain LYSX1. In vivo application of LYSX1 to tomato seedlings decreased the number of egg masses and galls and increased the root and shoot fresh weight. Treatment of half of the split-root system with LYSX1 reduced nematode penetration to the other half by 41.64%. Strain LYSX1 was identified as Bacillus halotolerans.

Conclusion

Bacillus halotolerans LYSX1 is a potential microbe for the sustainable biocontrol of root-knot nematodes through induced systemic resistance in tomato.

     

Effects of Inoculum Level and Time of Microcoleus vaginatus on Control of Meloidogyne incognita on Tomato

Application of Microcoleus vaginatus, a blue-green alga (Cyanobacterium) at different levels along with Meloidogyne incognita, second stage larvae, in the rhizosphere of tomato plants; showed that the plant growth as well as yield of tomato were increased and gall formations and nematode populations decreased with the increase in inoculum level of M. vaginatus. An inoculum level of 20 ml endospores suspension of M. vaginatus (2.4 × 10⁶ endospores per ml) per plant was optimum to reduce nematode attack with a population density of 1000 larvae per kg soil. Plant growth and yield of fruits were greatly suppressed and gall formations on roots, and nematode populations in soil were increased when M. incognita larvae added five days prior to M. vaginatus inoculation. On the other hand, when M. vaginatus inoculated ten days before nematode inoculation, suppressive effect of M. incognta on plants was reduced and their population density as well as gall formations were also decreased significantly. The efficacy of simultaneous inoculation of both nematode and M. vaginatus was lied in between two treatments discussed above.     

Interaction of Meloidogyne javanica with Different Races of Meloidogyne incognita

The interspecific interactions of Meloidogyne javanica with races 1, 2, 3, and 4 of M. incognita on tomato were determined. Impacts of the interactions on fecundity and morphometrics of females were also examined. Mutually inhibitory interactions occurred between M. javanica and the races of M. incognita, but the negative interactions did not reflect in plant growth. Numbers of root galls, egg masses, mature females, total population, fecundity, and reproduction factor declined in concomitant treatments, but the morphometrics of the females remained unaltered. In general, mutual suppressive effects in all parameters were smaller for M. javanica than M. incognita, but some variations occurred among the races of M. incognita. Race 2 appeared to be more competitive than other races. The interaction between the species was not intense; therefore, the species coexist in mixed populations in agricultural fields.     

Characterization of Anionic Peroxidases in Tomato Isolines Infected by Meloidogyne incognita

Changes in peroxidase activity during nematode infection were studied using root extracts of tomato near-isogenic lines differing in resistance to Meloidogyne incognita. Total peroxidase activity increased slightly in crude extracts of four susceptible isolines but doubled in two resistant lines, Monita and Motaci. Nematode infection enhanced levels of both p-phenylenediamine-pyrocatechol oxidase and syringaldazine oxidase 7 days after inoculation, especially in resistant lines. This elevated peroxidase activity in resistant isolines was caused by an increase in anionic peroxidase activity. These enzymes, which likely are involved in lignification, were isolated and purified from tomato isolines by ammonium sulfate precipitation, high performance ion-exchange chromatography, and gel electrophoresis. The purified anionic peroxidase extracts contained an electrophoretic band with Rf 0.51 that was present in extracts of infected but not uninfected roots.     

Biological Control of Meloidogyne hapla on Alfalfa and Tomato with the Fungus Meria coniospora

This study was to determine whether Arthrobotrys flagrans, A. oligospora, and Meria coniospora would control the root-knot nematode Meloidogyne hapla on alfalfa and tomato. Alfalfa seeds were coated with a fungus-rye powder in 2% cellulose and were planted in infested soil. Three-week-old seedlings from seed treated with M. coniospora had 60% and 58% fewer galls in two experiments than did seedlings from untreated seeds. Numbers of J2 in the soil were not reduced. Plant growth did not improve. When seed of tomato were coated with M. coniospora and planted in M. hapla-infested soil, roots had 34% fewer galls and 47% fewer J2 in the soil at 28 days. After 56 days there was no reduction in J2 numbers. Plant growth did not improve. When roots of tomato transplants were dusted with M. coniospora fungus-rye powder or sprayed with a spore suspension before planting in M. hapla-infested soil, 42% and 35%, respectively, fewer galls developed in 28 days on treated roots than on roots not treated with fungus. The numbers of J2 extracted from roots or recovered from soil were not reduced, however, and plant growth did not improve.     

The effect of host plants on Tetranychus evansi, Tetranychus urticae (Acari: Tetranychidae) and on their fungal pathogen Neozygites floridana (Entomophthorales: Neozygitaceae)

In a series of tritrophic-level interaction experiments, the effect of selected host plants of the spider mites, Tetranychus evansi and Tetranychus urticae, on Neozygites floridana was studied by evaluating the attachment of capilliconidia, presence of hyphal bodies in the infected mites, mortality from fungal infection, mummification and sporulation from fungus-killed mite cadavers. Host plants tested for T. evansi were tomato, cherry tomato, eggplant, nightshade, and pepper while host plants tested for T. urticae were strawberry, jack bean, cotton and Gerbera. Oviposition rate of the mites on each plant was determined to infer host plant suitability while host-switching determined antibiosis effect on fungal activity. T. evansi had a high oviposition on eggplant, tomato and nightshade but not on cherry tomato and pepper. T. urticae on jack bean resulted in a higher oviposition than on strawberry, cotton and Gerbera. Attachment of capilliconidia to the T. evansi body, presence of hyphal bodies in infected T. evansi and mortality from fungal infection were significantly higher on pepper, nightshade and tomato. The highest level of T. evansi mummification was observed on tomato. T. evansi cadavers from tomato and eggplant produced more primary conidia than those from cherry tomato, nightshade and pepper. Switching N. floridana infected T. evansi from one of five Solanaceous host plants to tomato had no prominent effect on N. floridana performance. For T. urticae, strawberry and jack bean provided the best N. floridana performance when considering all measured parameters. Strawberry also had the highest primary conidia production. This study shows that performance of N. floridana can vary with host plants and may be an important factor for the development of N. floridana epizootics.     

Morphological and Molecular Evaluation of a Meloidogyne hapla Population Damaging Coffee (Coffea arabica) in Maui,Hawaii

An unusual population of Meloidogyne hapla, earlier thought to be an undescribed species, was found causing large galls, without adventitious roots, and substantial damage to coffee in Maui, Hawaii. Only in Brazil had similar damage to coffee been reported by this species. Unlike M. exigua from South and Central America, this population reproduced well on coffee cv. Mokka and M. incognita-susceptible tomato but poorly on tomato with the Mi resistance gene. Characterization included SEM images, esterase isozymes, and five DNA sequences: i) the D3 segment of the large subunit (LSU-D3 or 28S) rDNA, ii) internal transcribed spacer (ITS-1) rDNA, iii) intergenic spacer (IGS) rDNA, iv) the mitochondrial interval from cytochrome oxidase (CO II) to 16S mtDNA, and v) the nuclear gene Hsp90. Sequences for ITS-1, IGS, and COII were similar to other M. hapla populations, but within species ITS-1 variability was not less than among species. One LSU-D3 haplotype was similar to a previously analyzed population with two minor haplotypes. Hsp90 exhibited some variation between Maryland and Hawaiian populations distinct from other species. Females were narrow with wide vulval slits, large interphasmidial distances, and more posterior excretory pores; 20% of perineal patterns had atypical perivulval lines. Males had a low b ratio (<12 µm). Juveniles had a short distance between stylet and dorsal gland orifice. Juvenile body length was short (<355 µm) and was different between summer and winter populations.     

Root Penetration by Meloidogyne incognita Juveniles Infected with Bacillus Penetrans

Bacillus penetrans inhibited penetration by Meloidogyne incognita second-stage juveniles (J2) into tomato roots in the laboratory and greenhouse. Spores from this Florida population of B. penetrans attached to J2 of M. javanica, M. incognita, and M. arenaria. A greater proportion of J2 of M. javanica were infected than were J2 of either M. incognita or M. arenaria, and a greater number of spores attached to M. incognita than to M. arenaria.     

Ethylene Production by Meloidogyne spp.-Infected Plants

Gall size and rates of ethylene production by various hosts infected with Meloidogyne javanica and by excised tomato root cultures infected with M. javanica or M. hapla were measured. Infection with M. javanica increased the rate of ethylene production in dicotyledonous plants (cabbage, pea, carrot, cucumber, carnation, and tomato), but not in infected monocotyledonous plants (corn, wheat, and onion). Nematode infection induced large galls on roots of dicotyledonous, but not monocotyledonous, plants. Excised tomato roots in culture infected with M. javanica produced ethylene at high rates and formed large galls, whereas roots infected with M. hapla produced ethylene at low rates and induced smaller galls.