A Strain of Bacillus thuringiensis for the Control of Plant‐parasitic Nematodes

An isolate of Bacillus thuringiensis, designated CR‐371, was evaluated for efficacy in controlling plant‐parasitic nematodes. This isolate was first shown to be nematicidal to Caenorhabditis elegans in an in vitro laboratory assay. Treatment resulted in a significant reduction in galls due to root‐knot nematode on tomato in a greenhouse trial. In two field trials in Puerto Rico, CR‐371‐treated tomatoes and pepper had significantly fewer root galls due to Meloidogyne incognita than untreated controls, and populations of Rotylenchulus reniformis were smaller. In one experiment, CR‐371 treatment was associated with significant increases in pepper yields, while in the second trial small yield increases of pepper and tomato occurred. In a greenhouse trial, incorporation of CR‐371 into a methyl cellulose seed coat gave similar control of root‐knot nematode on tomato as compared to CR‐371 applied as a drench. CR‐371‐treated strawberry plants also had smaller populations of Pratylenchus penetrans in roots in a greenhouse trial in Massachusetts.     

Banana Rhizodeposition: Characterization of Root Border Cell Production and Effects on Chemotaxis and Motility of the Parasitic Nematode Radopholus similis

Rhizodeposition was collected from root tips of banana (Musa acuminata). Two varieties, Grande naine and Yangambi km5, susceptible and resistant towards the burrowing nematode Radopholus similis, respectively, were examined for root border cell production under in vitro and in soil growing conditions. Two types of cells were observed in rhizodeposition: spherical cells containing large amyloplasts, called statocytes (8% of total), and long ellipsoidal border cells (92%). Border cell production was high, related to root length and not different between the two cultivars. Rhizodeposition from plants grown in soil contained similar amounts of border cells, but viability was lower than in in vitro grown plants. Chemotaxis and motility assays were performed to test the effects of banana roots, roots without rhizodeposition, rhizodeposition, exudates and border cells on the behaviour of R. similis. Roots of both the susceptible and resistant variety attracted R. similis, but only in the presence of rhizodeposition. Isolated rhizodeposition of Yangambi km5 also attracted nematodes. Border cells and exudates did not affect nematode chemotaxis. Roots of both varieties induced quiescence in R. similis in the chemotaxis assay. Thirty to fifty percent of the nematodes became temporary quiescent in the motility assay with rhizodeposition and exudates. The effect lasted for 24 h for Grande naine and up to 3 days for Yangambi km5. Rhizodeposition collected from plants grown in soil affected neither R. similis chemotaxis nor motility. Overall, there were no indications that rhizodeposition is involved in preformed resistance against R. similis in Yangambi km5.     

Expression of tomato salicylic acid (SA)‐responsive pathogenesis‐related genes in Mi‐1‐mediated and SA‐induced resistance to root‐knot nematodes

The expression pattern of pathogenesis‐related genes PR‐1, PR‐2 and PR‐5, considered as markers for salicylic acid (SA)‐dependent systemic acquired resistance (SAR), was examined in the roots and shoots of tomato plants pre‐treated with SA and subsequently infected with root‐knot nematodes (RKNs) (Meloidogyne incognita). PR‐1 was up‐regulated in both roots and shoots of SA‐treated plants, whereas the expression of PR‐5 was enhanced only in roots. The over‐expression of PR‐1 in the whole plant occurred as soon as 1 day after SA treatment. Up‐regulation of the PR‐1 gene was considered to be the main marker of SAR elicitation. One day after treatment, plants were inoculated with active juveniles (J2s) of M. incognita. The number of J2s that entered the roots and started to develop was significantly lower in SA‐treated than in untreated plants at 5 and 15 days after inoculation. The expression pattern of PR‐1, PR‐2 and PR‐5 was also examined in the roots and shoots of susceptible and Mi‐1‐carrying resistant tomato plants infected by RKNs. Nematode infection produced a down‐regulation of PR genes in both roots and shoots of SA‐treated and untreated plants, and in roots of Mi‐carrying resistant plants. Moreover, in resistant infected plants, PR gene expression, in particular PR‐1 gene expression, was highly induced in shoots. Thus, nematode infection was demonstrated to elicit SAR in shoots of resistant plants. The data presented in this study show that the repression of host defence SA signalling is associated with the successful development of RKNs, and that SA exogenously added as a soil drench is able to trigger a SAR‐like response to RKNs in tomato.     

Comparing simple root phenotyping methods on a core set of rice genotypes

Interest in belowground plant growth is increasing, especially in relation to arguments that shallow‐rooted cultivars are efficient at exploiting soil phosphorus while deep‐rooted ones will access water at depth. However, methods for assessing roots in large numbers of plants are diverse and direct comparisons of methods are rare. Three methods for measuring root growth traits were evaluated for utility in discriminating rice cultivars: soil‐filled rhizotrons, hydroponics and soil‐filled pots whose bottom was sealed with a non‐woven fabric (a potential method for assessing root penetration ability). A set of 38 rice genotypes including the OryzaSNP set of 20 cultivars, additional parents of mapping populations and products of marker‐assisted selection for root QTLs were assessed. A novel method of image analysis for assessing rooting angles from rhizotron photographs was employed. The non‐woven fabric was the easiest yet least discriminatory method, while the rhizotron was highly discriminatory and allowed the most traits to be measured but required more than three times the labour of the other methods. The hydroponics was both easy and discriminatory, allowed temporal measurements, but is most likely to suffer from artefacts. Image analysis of rhizotrons compared favourably to manual methods for discriminating between cultivars. Previous observations that cultivars from the indica subpopulation have shallower rooting angles than aus or japonica cultivars were confirmed in the rhizotrons, and indica and temperate japonicas had lower maximum root lengths in rhizotrons and hydroponics. It is concluded that rhizotrons are the preferred method for root screening, particularly since root angles can be assessed.     

Nematicidal activity of culture filtrate of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> against <Emphasis Type="Italic">Meloidogyne hapla</Emphasis>

Culture filtrates of Beauveria bassiana at different concentrations were evaluated for nematicidal activity against the northern root knot nematode (Meloidogyne hapla); bioassays included egg hatching, mortality and infectivity on tomato plants in pots under glasshouse conditions. The percentage mortality and inhibition of hatching of root-knot nematode were directly proportional to the concentration of culture filtrates of B. bassiana. Soil drenching with culture filtrate of B. bassiana significantly reduced nematode population densities in soil and in the roots and subsequent gall formation and egg-mass production by M. hapla under glasshouse conditions.     

The development of plant-parasitic nematode infestations on micro-propagated banana plants following field control measures in Côte d'Ivoire

Of the weeds that were found in banana production areas, only Asystasia gangetica was parasitised by Radopholus similis; Helicotylenchus multicinctus and Hoplolaimus pararobustus were able to parasitise all weed species. Field trials were carried out in Cote d'Ivoire to assess the potential for using nematode-free micro-propagated banana plants following cultural and chemical methods for nematode control. Banana (Musa acuminata) cv. Poyo was examined for nematodes after weed fallow, flooding and chemical treatment. Before replanting bananas, nematode assays showed that: i) all nematode species declined but were not eliminated after a 1,3-dichloropropene soil treatment; ii) H. multicinctus, H. pararobustus and Cephalenchus emarginatus were still present after either a 2-year weed fallow (dominated by Chromolaena odorata or Asystasia gangetica) or a 10-week flooding; iii) R. similis did not persist after fallowing or flooding. All nematode species were found in plots treated with 1,3-dichloropropene and that had been planted with bullheads, suckers or nematode-free micropropagated plants. After both fallow and flooding, R. similis was reintroduced on infested planting materials (bullheads and suckers) even when they were pre-treated with a nematicide. When fallow and flooded plots were planted with nematode-free materials (vitro-plants), R. similis did not appear in the roots for two vegetative cycles. The other species, still present in the soil, invaded the roots and increased slowly in numbers. These results were confirmed in commercial banana plantations.     

Screenhouse and Field Persistence of Nonpathogenic Endophytic Fusarium oxysporum in Musa Tissue Culture Plants

Two major biotic constraints to highland cooking banana (Musa spp., genome group AAA-EA) production in Uganda are the banana weevil Cosmopolites sordidus and the burrowing nematode Radopholus similis. Endophytic Fusarium oxysporum strains inoculated into tissue culture banana plantlets have shown control of the banana weevil and the nematode. We conducted screenhouse and field experiments to investigate persistence in the roots and rhizome of two endophytic Fusarium oxysporum strains, V2w2 and III4w1, inoculated into tissue-culture banana plantlets of highland cooking banana cultivars Kibuzi and Nabusa. Re-isolation of F. oxysporum showed that endophyte colonization decreased faster from the rhizomes than from the roots of inoculated plants, both in the screenhouse and in the field. Whereas rhizome colonization by F. oxysporum decreased in the screenhouse (4–16 weeks after inoculation), root colonization did not. However, in the field (17–33 weeks after inoculation), a decrease was observed in both rhizome and root colonization. The results show a better persistence in the roots than rhizomes of endophytic F. oxysporum strains V2w2 and III4w1.     

Dual inoculation of Fusarium oxysporum endophytes in banana: effect on plant colonization,growth and control of the root burrowing nematode and the banana weevil

The burrowing nematode (Radopholus similis (Cobb) Thorne) and the banana weevil (Cosmopolites sordidus Germar, Coleoptera: Curculionidae) are major pests of banana (Musa spp.) in the Lake Victoria basin region of Uganda. Among biological options to control the two pests is the use of non-pathogenic Fusarium oxysporum Schltdl.: Fries endophytes of banana. We investigated the ability of endophytic F. oxysporum isolates Emb2.4o and V5w2 to control the banana weevil and the burrowing nematode, alone and in combination. Plant colonization by the endophytes was determined by inoculating their chemical-resistant mutants separately and in combination, onto banana roots. Plant growth promotion was determined by measuring plant height, girth, number of live roots and fresh root weight at harvest, and control of the nematode and weevil was determined by challenging endophyte-inoculated plants with the pests 8 weeks after endophyte inoculation. Endophytic root colonization was highest in plants inoculated with both endophytes, compared with those inoculated with only one of the endophytes. Root colonization was better for isolate V5w2 than Emb2.4o. Dually inoculated plants showed a significant increase in height, girth, fresh root weight and number of functional roots following nematode challenge. Nematode numbers in roots were reduced 12 weeks after challenge of 8-week-old endophyte-inoculated plants. Significant reductions in weevil damage were observed in the rhizome periphery, inner and outer rhizomes, compared with endophyte non-inoculated controls. We conclude that dual inoculation of bananas with endophytic isolates Emb2.4o and V5w2 increases root colonization by the endophytes, reduces nematode numbers and weevil damage, and enhances plant growth in the presence of nematode infestation.     

Effect of Conditioning Treatments on the Survival of Radopholus similis at High Temperatures

Heat treatments are an environmentally safe method for eliminating quarantine pests from tropical foliage. Conditioning heat treatments can induce thermotolerance against subsequent and otherwise phytotoxic temperatures in tropical foliage, allowing heat treatments to be even more effective. However, if thermotolerance is also induced in nematodes of quarantine significance like Radopholus similis, heat treatments would be rendered ineffective. A lethal thermal death point (LT_99.9) was established for R. similis by recording mortality at 25 (control temperature), 43°C, 45°C, 47°C, or 49°C after a 0, 1-, 2-, 4-, 6-, 8-, 10-, 12-, or 15-minute exposure. In a second experiment, nematodes were conditioned at 35, 40, or 45°C for 0, 15, 30, 60, 120, and 180 minutes, allowed to rest for 3 hours, and then challenged at 47°C for 5 minutes. No nematodes survived the challenge heat treatment; rather, nematode mortality was hastened by the conditioning treatment itself. In a third experiment, R. similis inside anthurium roots were conditioned at 25°C or 40°C for 15 minutes and then treated at 45°C for up to 8 minutes. Mortality of conditioned and unconditioned nematodes was similar (P > 0.1). Conditioning treatments increase plant thermotolerance but do not induce thermotolerance in R. similis. Heat treatments have promise as disinfection protocols for quarantines.     

Characterization of a New Burrowing Nematode Population,Radopholus citrophilus,from Hawaii

Karyotype, host preference, isozyzme patterns, morphometrics, and mating behavior of two burrowing nematode populations from Hawaii, one infecting Anthurium sp. and the second infecting Musa sp., were compared with Radopholus similis and R. citrophilus populations from Florida. The population from Anthurium sp. had five chromosomes (n = 5), and that from Musa sp. had four (n = 4). Neither of the Hawaiian nematode populations persisted in roots of Citrus limon or C. aurantium. Anthurium clarinerivum and A. hookeri were hosts of the burrowing nematode population from anthurium in Hawaii and of R. citrophilus from Florida, whereas the two anthurium species were poor hosts of the population from Musa sp. in Hawaii and R. similis from Florida. The isozyme pattern of the population isolated from anthurium was identical to that of R. citrophigus, whereas the pattern of the population from banana in Hawaii was identical to that of R. similis. Mating behavior between the burrowing nematode population isolated from Anthurium sp. and a Florida population of R. citrophilus supports their close taxonomic relationship. Mating was observed between the population from Anthurium sp. and the Florida population of R. citrophilus but not between the Hawaiian burrowing nematode population isolated from Musa sp. and a Florida population of R. citrophilus. These findings indicate that a previously unidentified population of R. citrophilus which does not parasitize citrus occurs in Hawaii.     

Über die Kallosebildung in Siebröhren von Kartoffelpflanzen in Abhängigkeit von Blattrollvirusinfektion und chemisch-physikalischen Bedingungen

All the cultivars of tomato tested were found to be infected with the root‐knot nematode, Meloidogyne incognita, however, to varying extent. There was also reducton in plant growth parameters accordingly. Further reduction in plant growth of tomato cultivars was recorded when plants also received the treatment with the heavy metal, lead, notwithstanding the fact that the presence of lead also reduced the root‐knot development. Tomato cv. Arka Vikas showed minimum reduction in plant growth with single or combined treatment with the nematode and the heavy metal.     

Effects of Thermotherapy Treatments on Systemic Agrobacterium vitis in Dormant Grape Cuttings

Agrobacterium tumefaciens biovar 3 (A. vitis) was eradicated or reduced below the level of detection in dormant cuttings of grape [Vitis vinifera cultivar (cv.) Thompson seedless] and rootstock NAZ3 (V. vinifera × V. rupestris) by hot‐water treatment (exposure to 50°C for 30 min). Strains of A. vitis varied in their sensitivity to heat, but were generally more sensitive than strains of A. tumefaciens biovar 1 or biovar 2. Populations of about 10³ CFU/ml in broth were killed by a 30‐min treatment. Biovar 1 strains were apparently unaffected by 50°C, even when exposed for 30 min. Non‐tumorigenic biovar 1 strains were recovered from hot‐water‐treated cuttings. An assessment was also made of the effect of treatment on growth parameters of cuttings in a field nursery after 9 months. The effect of hot‐water treatment on the vitality and growth of vines varied with different cultivars or rootstocks. The number and length of canes, root dry weight and length and diameter of trunks, increased in most instances. Treatment and time usually did affect bud survival and, in most cases, increased the level of callus formation at the base of the cuttings. Hot‐water treatment may offer a simple, effective, economical and environmentally safe means of eradicating A. vitis from dormant grape cuttings.     

Ralstonia solanacearum colonization of tomato roots infected by Meloidogyne incognita

Bacterial wilt, caused by Ralstonia solanacearum, is one of the most serious diseases of tomato (Solanum lycopersicum). Concomitant infection of R. solanacearum and root‐knot nematode Meloidogyne incognita increases the severity of bacterial wilt in tomato, but the role of this nematode in disease complexes involving bacterial pathogens is not completely elucidated. Although root wounding by root‐knot nematode infection seems to play an important role, it might not entirely explain the increased susceptibility of plants to R. solanacearum. In the present study, green fluorescent protein (GFP)‐labelled R. solanacearum distribution was observed in the root systems of the tomato cultivar Momotaro preinoculated with root‐knot nematode or mock‐inoculated with tap water. Fluorescence microscopy revealed that GFP‐labelled R. solanacearum mainly colonized root‐knot nematode galls, and little or no green fluorescence was observed in nematode‐uninfected roots. These results suggest that the gall induced by the nematode is a suitable location for the growth of R. solanacearum. Thus, it is crucial to control both R. solanacearum and root‐knot nematode in tomato production fields to reduce bacterial wilt disease incidence and effects.     

Pre-plant bacterisation: a strategy for delivery of beneficial endophytic bacteria and production of disease-free plantlets of black pepper (Pipernigrum L.)

Naturally occurring endophytic bacteria from black pepper vines were found to exhibit strong antagonistic activities against Phytophthora capsici and Radopholus similis. In order to deliver these bacterial strains, as well as to produce disease-free plantlets of black pepper, a pre-plant stem and root bacterisation was standardised. Stem bacterisation with endophytic Pseudomonas spp. was found to suppress P. capsici infection (over 90% reduction in lesion length) on cut shoots. Pre-plant root bacterisation with Pseudomonas aeruginosa, Pseudomonas putida and Bacillus megaterium yielded over 60% of plantlets free from P. capsici infection on roots. Curtobacterium luteum and B. megaterium recorded over 70% reduction of nematode population in soil with concomitant production of over 65% of nematode-free plantlets. Besides protecting the plants from the pathogens, the bacteria were also found to enhance the growth of rooted cuttings. The biocontrol potential of the above endophytic bacteria and their exploitation for disease management in the black pepper nursery are discussed.     

Relative mycorrhizal dependency and mycorrhiza-nematode interaction in banana cultivars (Musa spp.) differing in nematode susceptibility

Four Musa cultivars, differing in nematode susceptibility, were selected to study their relative mycorrhizal dependency and to study the interaction between the arbuscular mycorrhizal fungus (AMF), Glomus mosseae, and two migratory endoparasitic nematodes, Radopholus similis and Pratylenchus coffeae. Mycorrhization with G. mosseae resulted in significantly better plant growth, even in the presence of R. similis and P. coffeae. No differences in relative mycorrhizal dependency (RMD) were observed among the four cultivars. G. mosseae suppressed nematode population build-up in Grande Naine and Pisang Jari Buaya. Only in the case of R. similis (Indonesian population) in Pisang Jari Buaya, no significant suppression was observed. In the case of P. coffeae, the AMF reduced the damage in the roots, caused by the nematodes. For R. similis, no reduction of damage was observed. In all, except one experiment, the frequency of the mycorrhizal colonisation was negatively affected by the nematodes.     

Effects of Glomus intraradices on the reproduction of the burrowing nematode (Radopholus similis) in dixenic culture

The effects of Glomus intraradices on the reproduction of the burrowing nematode Radopholus similis were studied under dixenic culture conditions. The life cycles of both the arbuscular mycorrhizal fungus (AMF) and the nematode were completed in presence of each other and a transformed carrot root as host. The AMF suppressed the R. similis population by almost 50% and thus increased protection of the root against the nematode. This reduction was significant for both females and males within roots. There was no correlation between nematode population density and either AMF internal root colonization, external hyphal development or spore production. These results demonstrate that the dixenic system, although artificial, is a valuable tool for studying AMF–nematode interactions, complementing the classical experimental approaches. Accepted: 6 February 2001     

Developmental and behavioural effects of the endophytic Fusarium moniliforme Fe14 towards Meloidogyne graminicola in rice

The root‐knot nematode, Meloidogyne graminicola, is an important pest of rice in many rice production areas worldwide. The endophyte Fusarium moniliforme strain Fe14, isolated from a disinfected root of rice, has previously shown potential antagonistic activity against M. graminicola. This study shows the effects of Fe14 on M. graminicola behaviour, infection, development and reproduction. The endophyte Fe14 colonisation significantly reduced M. graminicola penetration into rice roots by 55% and increased the male to female ratio nine times. The endophyte also delayed juvenile development into female inside the rice root. These results suggest a suboptimal performance of the giant cell and a cumulative effect of the endophyte on the long‐term root‐knot nematode population development. In split‐root assays, the application of Fe14 at the inducer side significantly reduced nematode invasion at the responder side by 38% and 60% in two independent trials. This result suggests a systemic effect of the endophyte on rice plants. The root exudates from Fe14‐treated plants were either less attractive or had repellent effect on nematode movement. The results, when compared to what was described for other endophytic Fusarium against other nematode species, may indicate a basal response mechanism initiated in the plant by endophytic Fusarium spp. The present study may give leads for unravelling the molecular mechanisms responsible for the induced systemic defence responses in plants.     

Hot water immersion of Allium hookeri roots for the control of the plant parasitic nematodes Meloidogyne javanica and Pratylenchus coffeae

Nematodes are important quarantine pests of bulbous plants such as hooker chives. Although control methods such as fumigation, chemical immersion, and heat are often applied, it has proved difficult to disinfect nematodes from plant roots in quarantine. As heat treatment has been successfully useful for the control of nematodes in other agricultural products in quarantine, we investigated the susceptibility and mortality rates of Meloidogyne javanica and Pratylenchus coffeae, which infest hooker chive roots, using a hot water immersion method. Heat damage to the hooker chive roots was noticeable at temperatures over 50°C. Temperatures for the effective time to kill 99% at 1 min (ET99) for M. javanica and P. coffeae juveniles were 49.3°C and 49.1°C, respectively. However, the time to kill 99% of M. javanica eggs at 48°C and 49°C were 27.0 min and 8.3 min, respectively. Using a thermal equilibrium formula, the optimum commercial scale condition, in a 1400‐L chamber, for nematode control without associated plant damage was water immersion at 48.2°C for 30 min or at 49.2°C for 13 min with a filling ratio less than 12%. This result can be applicable for the nematode disinfestation of hooker chive roots in plant quarantine.