Artificial inoculation of banana tissue culture plantlets with indigenous endophytes originally derived from native banana plants

Fusarium wilt disease of banana is one of the most harmful fungal diseases affecting banana production worldwide. We hypothetically proposed that the loss of indigenous endophytes in tissue culture propagation of banana might be related to increased disease severity on banana plants. In the present study, a mixture of uncultivated endophytes, which was originally derived from native healthy banana plant in plantation, was used to artificially inoculate banana tissue culture plantlets. A broad spectrum of bacterial communities was detected in the roots of artificially inoculated plantlets by 16S ribosomal RNA gene analysis, and γ-Proteobacteria was identified as the dominant group. Banana wilt pathogen Fusarium oxysporum f. sp. cubense race 4 was inoculated to the plantlets after potting to investigate disease progress. With early diagnosis of fungal pathogen infection, 54% reduction was detected in artificially inoculated plantlets compared to endophyte-free control plantlets. The re-introduction of naturally-occurring endophytes into tissue culture banana plantlets led to a 67% suppression rate of wilt disease at the fifth month after pathogen infection on plantlets in the greenhouse. In addition to disease suppression, growth of host plantlets was also promoted with the inoculation of endophytes. The artificial inoculation method provided a foundational understanding of ecological enrichment to control banana wilt disease in future.     

Potential of Foliar,Dip, and Injection Applications of Avermectins for Control of Plant-Parasitic Nematodes

Studies were conducted to determine the potential of two avermectin compounds, abamectin and emamectin benzoate, for controlling plant-parasitic nematodes when applied by three methods: foliar spray, root dip, and pseudostem injection. Experiments were conducted against Meloidogyne incognita on tomato, M. javanica on banana, and Radopholus similis on banana. Foliar applications of both avermectins to banana and tomato were not effective for controlling any of the nematodes evaluated. Root dips of banana and tomato were moderately effective for controlling M. incognita on tomato and R. similis on banana. Injections (1 ml) of avermectins into banana pseudostems were effective for controlling M. javanica and R similis, and were comparable to control achieved with a conventional chemical nematicide, fenamiphos. Injections of 125 to 2,000 μg/plant effectively controlled one or both nematodes on banana; abamectin was more effective than emamectin benzoate for controlling nematodes.     

Non-pathogenic Fusarium oxysporum endophytes provide field control of nematodes,improving yield of banana (Musa sp.)

Endophytic colonization by the fungus Fusarium oxysporum can result in increased host resistance to pests and diseases, and greater biomass production. However, few studies have assessed the field performance of this fungus for biological control of pests and diseases in banana. Further to greenhouse assessment, studies were carried out to evaluate the performance of F. oxysporum strains against plant-parasitic nematodes on banana (Musa sp., cv. Giant Cavendish and cv. Grand Nain) in the field using tissue-cultured plants. Plants were inoculated separately with one of three strains (V5W2, Eny 7.11o and Emb 2.4o) before being inoculated with Pratylenchus goodeyi and Helicotylenchus multicinctus in an on-station trial and in an on-farm trial planted in a field naturally infested with the same nematodes. All three endophytic strains significantly suppressed P. goodeyi and H. multicinctus densities and damage in the field. On-station, nematode population densities were reduced by >45% in endophyte-inoculated plants compared to non-inoculated plants, while percentage root necrosis was reduced by >20%. Similarly, on-farm, nematode damage to roots and densities were also significantly lower in endophyte-inoculated plants compared with control plants. Significantly improved yields were observed for plants inoculated with endophytes when compared to the control plants, with inoculation with strains Emb 2.4o and V5W2 resulting in up to 35% and 36% increased banana yields, respectively, for the on-station trial. For the on-farm trial, up to 20% increase in yields were observed for strain Eny 7.11o compared to control plants. This study provides the first report from the field in Africa on the reduction of nematode populations and damage, and the increase in banana production by fungal endophytes. The study shows that endophytes have potential to enhance yields of tissue-cultured banana plants and protect them against pests.     

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.     

Aggressiveness and Damage Potential of Central American and Caribbean Populations of Radopholus spp. in Banana

Monoxenic cultures of burrowing nematode populations extracted from banana roots from Belize, Guatemala, Honduras, and Costa Rica were established on carrot discs. Cultures of Radopholus spp. were also obtained from Florida, Puerto Rico, Dominican Republic, and Ivory Coast. The aggressiveness (defined as reproductive fitness and root necrosis) of these populations was evaluated by inoculating banana plants (Musa AAA, cv. Grande Naine) with 200 nematodes/plant. Banana plants produced by tissue culture were grown in 0.4-liter styrofoam cups, containing a 1:1 mix of a coarse and a fine sand, at ca. 27 °C and 80% RH. Banana plants were acclimated and allowed to grow for 4 weeks prior to inoculation. Plant height, fresh shoot and root weights, root necrosis, and nematode population densities were determined 8 weeks after inoculation. Burrowing-nematode populations varied in aggressiveness, and their reproductive fitness was generally related to damage reported in the field. Plant height and fresh shoot and root weight did not reflect damage caused by nematodes under our experimental conditions. Necrosis of primary roots was closely related to the reproductive fitness of the nematode populations. Variation in aggressiveness among nematode populations followed a similar trend in the two susceptible hosts tested, Grande Naine and Pisang mas. All nematode populations had a low reproductive factor (Rf ≤2.5) in the resistant host except for the Ivory Coast population which had a moderate reproductive factor (Rf ≤ 5) on Pisang Jari Buaya. This is the first report of a burrowing nematode population parasitizing this important source of resistance to R. similis.     

Morphological Differences Between Radopholus citrophilus and R. similis

SEM observations of the external morphology of populations of Radopholus citrophilus and R. similis revealed several diagnostic differences. The cloaco-spicular orifice on males of R. citrophilus had three to seven genital papillae (anterior hypoptygmata), whereas males of R. similis were either smooth or had one or two shorter genital papillae (anterior hypoptygmata). Females of R. citrophilus had four annules in the region of the vulval opening, but R. similis had five annules in the same region. The labial disc and lateral lips appeared to be of diagnostic significance, but these areas were more susceptible to artifacts due to fixation. An unknown population of Radopholus from Puerto Rico with a chromosome number of n = 4 was morphologically similar to R. similis. These morphological differences provide additional support that R. citrophilus and R. similis are distinct species.     

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.     

Biological control of Radopholus similis in banana by combined application of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus

The biological control efficacy of single or multiple applications of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus toward Radopholus similis was investigated in pot trials with banana under glasshouse conditions. R. similis was controlled substantially in single and combined applications of F. oxysporum with P. lilacinus or B. firmus. The combination of F. oxysporum and P. lilacinus caused a 68.5% reduction in nematode density whereas the individual applications reduced the density by 27.8% and 54.8% over the controls, respectively. Combined application of F. oxysporum and B. firmus was the most effective treatment in controlling R. similis on banana (86.2%), followed by B. firmus alone (63.7%). The compatibility of the biocontrol agents, as well the capacity of F. oxysporum to colonize banana roots in the absence or presence of P. lilacinus was also investigated. P. lilacinus did not adversely affect endophytic colonization by F. oxysporum. Biological control of R. similis in banana can therefore be enhanced via combined applications of antagonists with different modes of action that target different stages in the infection process. Handling editor: Ralf-Udo Ehlers     

Endophytic microorganisms as potential growth promoters of banana

The potential of endophytic microorganisms in promoting the growth of their host plant was determined by artificially introducing five isolates (bacterial and fungal strains: UPM31F4, UPM31P1, UPM14B1, UPM13B8, UPM39B3) isolated from the roots of wild bananas into both healthy and diseased banana plantlets (Berangan cv. Intan). The response of the host plants to endophytic infection was assessed by measuring the change in four growth parameters: plant height, pseudostem diameter, root mass and total number of leaves. The endophytes tested as growth promoters were found to have a significant effect in both healthy and Fusarium-infected (diseased) plantlets. In both experimental systems, the bacterial isolate UPM39B3 (Serratia) and fungal isolate UPM31P1 (Fusarium oxysporum) showed promising growth-promoting properties. Isolate UPM39B3 (Serratia) induced the largest increases in all four growth parameters in healthy plantlets – 3.14 cm (height), 1.12 cm (pseudostem diameter), 2.12 g (root mass) and 1.12 (total number of leaves plant⁻¹) – followed by isolate UPM31P1 (Fusarium oxysporum). The beneficial effect of UPM39B3 (Serratia) and UPM31P1 (Fusarium oxysporum) was also reflected in the diseased plantlets, where pre-treatments with the isolates either singly (T6: UPM31P1; T8: UPM39B3) or in a mixture (T7: UPM31P1 + UPM39B3; T9: UPM14B1 + UPM13B8 + UPM39B3) were able to sustain the growth of plantlets, with significantly higher growth values than those in diseased plantlets that were not infected with endophytes (T10: FocR4). These results demonstrate the economic significance of these endophytic isolates, particularly UPM39B3 (Serratia) and UPM31P1 (Fusarium oxysporum), both as potential growth promoters of banana and as agents rendering tolerance towards Fusarium wilt as a strategy in the management of Fusarium wilt of banana via improved vegetative growth.     

Population Densities of Five Migratory Endoparasitic Nematodes in Carrot Disk Cultures

Numbers of nematodes recovered per culture varied greatly among five species cultured on carrot disks. Radopholus similis and Pratylenchus vulnus showed the highest population densities, with 23,400-fold and 16,600-fold increases, respectively, in 90 days. Final populations of P. thornei and Zygotytenchus guevarai were similar but lower than those of R. similis and P. vulnus. The population of P. neglectus increased 74 times. Species with the greatest reproduction in this study reproduce sexually.     

Association of Criconemella xenoplax and Fusarium spp. with Root Necrosis and Growth of Peach

Criconemella xenoplax, Fusarium solani, and F. oxysporum caused necrosis of Nemaguard peach feeder roots in greenhouse tests. Root necrosis was more extensive in the presence of either fungus than wtih C. xenoplax alone. Shoot growth and plant height were less for plants inoculated with F. oxysporum or F. solani than for plants inoculated with the fungi plus C. xenoplax. Neither synergistic nor additive effects on root necrosis or plant growth occurred between C. xenoplax and the fungal pathogens.     

Predisposition of Broadleaf Tobacco to Fusarium Wilt by Early Infection with Globodera tabacum tabacum or Meloidogyne hapla

In greenhouse experiments, broadleaf tobacco plants were inoculated with tobacco cyst (Globodera tabacum tabacum) or root-knot (Meloidogyne hapla) nematodes 3, 2, or 1 week before or at the same time as Fusarium oxysporum. Plants infected with nematodes prior to fungal inoculation had greater Fusarium wilt incidence and severity than those simultaneously inoculated. G. t. tabacum increased wilt incidence and severity more than did M. hapla. Mechanical root wounding within 1 week of F. oxysporum inoculation increased wilt severity. In field experiments, early-season G. t. tabacum control by preplant soil application of oxamyl indirectly limited the incidence and severity of wilt. Wilt incidence was 48%, 23%, and 8% in 1989 and 64%, 60%, and 19% in 1990 for 0.0, 2.2, and 6.7 kg oxamyl/ha, respectively. Early infection of tobacco by G. t. tabacum predisposed broadleaf tobacco to wilt by F. oxysporum.     

Improved Nematode Extraction from Carrot Disk Culture

Radopholus spp. were reared in carrot tissue culture via established procedures, with slight modification. Several plant tissue maceration enzymes and flotation media (salts and sucrose) were evaluated with regard to nematode toxicity and extraction efficiency. Best extraction of viable nematodes and eggs was attained when carrot tissue infested with Radopholus citrophilus or R. similis was macerated with a mixture of 0.50% driselase and 0.50% cellulysin, w/v each, with 2.5 ml of enzyme solution based for each gram of carrot tissue. Maceration slurries containing carrot tissue and nematodes were maintained in open flasks on a rotary shaker (175 rpm) at 26 C for 24 hours. Nematodes and eggs were extracted from resultant culture slurries by flotation with MgSO₄-7H₂0 (sp gr 1.1). A protocol is presented to extract large quantities of viable burrowing nematodes and their eggs from carrot disk cultures.     

Loline alkaloid production by fungal endophytes of Fescue species select for particular epiphytic bacterial microflora

The leaves of fescue grasses are protected from herbivores by the production of loline alkaloids by the mutualist fungal endophytes Neotyphodium sp. or Epichloë sp. Most bacteria that reside on the leaf surface of such grasses can consume these defensive chemicals. Loline-consuming bacteria are rare on the leaves of other plant species. Several bacterial species including Burkholderia ambifaria recovered from tall fescue could use N-formyl loline as a sole carbon and nitrogen source in culture and achieved population sizes that were about eightfold higher when inoculated onto plants harboring loline-producing fungal endophytes than on plants lacking such endophytes or which were colonized by fungal variants incapable of loline production. In contrast, mutants of B. ambifaria and other bacterial species incapable of loline catabolism achieved similarly low population sizes on tall fescue colonized by loline-producing Neotyphodium sp. and on plants lacking this endophytic fungus. Lolines that are released onto the surface of plants benefiting from a fungal mutualism thus appear to be a major resource that can be exploited by epiphytic bacteria, thereby driving the establishment of a characteristic bacterial community on such plants.     

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.     

Systemic acquired resistance in Cavendish banana induced by infection with an incompatible strain of Fusarium oxysporum f. sp. cubense

Fusarium wilt of banana is caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). The fact that there are no economically viable biological, chemical, or cultural measures of controlling the disease in an infected field leads to search for alternative strategies involving activation of the plant's innate defense system. The mechanisms underlying systemic acquired resistance (SAR) are much less understood in monocots than in dicots. Since systemic protection of plants by attenuated or avirulent pathogens is a typical SAR response, the establishment of a biologically induced SAR model in banana is helpful to investigate the mechanism of SAR to Fusarium wilt. This paper described one such model using incompatible Foc race 1 to induce resistance against Foc tropical race 4 in an in vitro pathosystem. Consistent with the observation that the SAR provided the highest level of protection when the time interval between primary infection and challenge inoculation was 10 d, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL, EC 4.3.1.5), peroxidase (POD, EC 1.11.1.7), polyphenol oxidase (PPO, EC 1.14.18.1), and superoxide dismutase (SOD, EC 1.15.1.1) in systemic tissues also reached the maximum level and were 2.00–2.43 times higher than that of the corresponding controls on the tenth day. The total salicylic acid (SA) content in roots of banana plantlets increased from about 1 to more than 5 μg g⁻¹ FW after the second leaf being inoculated with Foc race 1. The systemic up-regulation of MaNPR1A and MaNPR1B was followed by the second up-regulation of PR-1 and PR-3. Although SA and jasmonic acid (JA)/ethylene (ET) signaling are mostly antagonistic, systemic expression of PR genes regulated by different signaling pathways were simultaneously up-regulated after primary infection, indicating that both pathways are involved in the activation of the SAR.     

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.     

Comparison of Pratylenchus penetrans Infection and Maladera castanea Feeding on Strawberry Root Rot

The interaction of lesion nematodes, black root rot disease caused by Rhizoctonia fragariae, and root damage caused by feeding of the scarab larva, Maladera castanea, was determined in greenhouse studies. Averaged over all experiments after 12 weeks, root weight was reduced 13% by R. fragariae and 20% by M. castanea. The percentage of the root system affected by root rot was increased by inoculation with either R. fragariae (35% more disease) or P. penetrans (50% more disease) but was unaffected by M. castanea. Rhizoctonia fragariae was isolated from 9.2% of the root segments from plants not inoculated with R. fragariae. The percentage of R. fragariae-infected root segments was increased 3.6-fold by inoculation with R. fragariae on rye seeds. The presence of P. penetrans also increased R. fragariae root infection. The type of injury to root systems was important in determining whether roots were invaded by R. fragariae and increased the severity of black root rot. Pratylenchus penetrans increased R. fragariae infection and the severity of black root rot. Traumatic cutting action by Asiatic garden beetle did not increase root infection or root disease by R. fragariae. Both insects and diseases need to be managed to extend the productive life of perennial strawberry plantings.     

Pathological Relationship of Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis on alfalfa

Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis synergistically affected the mortality and plant growth of Ranger alfalfa, a cultivar susceptible to stem nematode and Fusarium wilt. The nematode-fungus relationship had an additive effect on mortality and plant growth of Lahontan (nematode resistant and Fusarium wilt susceptible) and of Moapa 69 (nematode susceptible and Fusarium wilt resistant). Mortality rates were 13, 16, 46, and 49% for Ranger; 4, 18, 26, and 28% for Lahontan; and 19, 10, 32, and 30% for Moapa 69 inoculated with D. dipsaci, F. oxysporum f. sp. medicaginis, and simultaneously and sequentially with D. dipsaci and F. oxysporum f. sp. medicaginis, respectively. Shoot weights as a percentage of uninoculated controls for the same treatments were 52, 84, 26, and 28%, for Ranger; 74, 86, 64, and 64% for Lahontan; and 50, 95, 44, and 39% for Moapa 69. Plant growth suppression was related to vascular bundle infection and discoloration of alfalfa root tissue. Disease severity and plant growth of alfalfa did not differ with simultaneous or sequential inoculations of the two pathogens. Fusarium oxysporum f. sp. medicaginis affected alfalfa growth but not nematode reproduction.     

Molecular and Biochemical Diversity Among Isolates of Radopholus spp. from Different Areas of the World

Eleven isolates of Radopholus similis from various banana-growing areas around the world and one isolate of R. bridgei from turmeric in Indonesia were compared using DNA and isoenzyme analysis. The polymerase chain reaction (PCR) was used to amplify a fragment of ribosomal DNA (rDNA), comprising the two internal transcribed spacers (ITS) and the 5.8S gene. Restriction fragment length polymorphisms (RFLPs) in this rDNA fragment were used to compare the 10 isolates. The analysis of this rDNA region revealed little variation among the isolates tested. However, data also were obtained by random amplified polymorphic DNA (RAPD) analysis of total DNA, and a hierarchical cluster analysis of these data arranged the R. similis isolates into two clusters. The first cluster consisted of isolates from Nigeria, Cameroon, Queensland, and Costa Rica; the second was comprised of isolates from Guinea, Guadeloupe, the Ivory Coast, Uganda, and Sri Lanka. The isolate of R. bridgei from turmeric in Indonesia appeared to be more divergent. This grouping was consistent with that obtained when phosphate glucose isomerase (PGI) isoenzyme patterns were used to compare the R. similis isolates. The results from both RAPD analysis and PGI isoenzyme studies indicate that two gene pools might exist within the R. similis isolates studied. No correlation could be detected between the genomic diversity as determined by RAPD analysis and either geographic distribution of the isolates or differences in their pathogenicity. The results support the hypothesis that R. similis isolates have been spread with banana-planting material.