Nuclear magnetic resonance: a tool for imaging belowground damage caused by Heterodera schachtii and Rhizoctonia solani on sugar beet.

Belowground symptoms of sugar beet caused by the beet cyst nematode (BCN) Heterodera schachtii include the development of compensatory secondary roots and beet deformity, which, thus far, could only be assessed by destructively removing the entire root systems from the soil. Similarly, the symptoms of Rhizoctonia crown and root rot (RCRR) caused by infections of the soil-borne basidiomycete Rhizoctonia solani require the same invasive approach for identification. Here nuclear magnetic resonance imaging (MRI) was used for the non-invasive detection of belowground symptoms caused by BCN and/or RCRR on sugar beet. Excessive lateral root development and beet deformation of plants infected by BCN was obvious 28 days after inoculation (dai) on MRI images when compared with non-infected plants. Three-dimensional images recorded at 56 dai showed BCN cysts attached to the roots in the soil. RCRR was visualized by a lower intensity of the MRI signal at sites where rotting occurred. The disease complex of both organisms together resulted in RCRR development at the site of nematode penetration. Damage analysis of sugar beet plants inoculated with both pathogens indicated a synergistic relationship, which may result from direct and indirect interactions. Nuclear MRI of plants may provide valuable, new insight into the development of pathogens infecting plants below- and aboveground because of its non-destructive nature and the sufficiently high spatial resolution of the method.

Systemically induced resistance and microbial competitive exclusion: implications on biological control.

The root-knot nematode, Meloidogyne incognita, is among the most damaging agricultural pests, particularly to tomato. The mutualistic endophytes Fusarium oxysporum strain Fo162 (Fo162) and Rhizobium etli strain G12 (G12) have been shown to systemically induce resistance toward M. incognita. By using triple-split-root tomato plants, spatially separated but simultaneous inoculation of both endophytes did not lead to additive reductions in M. incognita infection. More importantly, spatially separated inoculation of Fo162 and G12 led to a reduction in Fo162 root colonization of 35 and 39% when G12 was inoculated on a separate root section of the same plant in two independent experiments. In an additional split-root experiment, spatial separation of Fo162 and G12 resulted in a reduction of Fo162 root colonization of approximately 50% over the water controls in two independent experiments. The results suggested that the suppressive activity of G12 on Fo162 and M. incognita is possibly related to the induction of specific plant defense mechanisms. Thus, although Fo162 and G12 have the ability to systemically repress M. incognita infection in tomato, they can be considered incompatible biocontrol agents when both organisms are present simultaneously on the same root system.

Effect of Meloidogyne incognita inoculum density and application rate of Paecilomyces lilacinus strain 251 on biocontrol efficacy and colonization of egg masses analyzed by real-time quantitative PCR.

The fungal biocontrol agent, Paecilomyces lilacinus strain 251 (PL251), was evaluated for its potential to control the root-knot nematode Meloidogyne incognita on tomato at varying application rates and inoculum densities. Conversely to previous studies, significant dose-response relationships could not be established. However, we demonstrated that a preplanting soil treatment with the lowest dose of commercially formulated PL251 (2 × 10(5) CFU/g soil) was already sufficient to reduce root galling by 45% and number of egg masses by 69% when averaged over inoculum densities of 100 to 1,600 eggs and infective juveniles per 100 cm(3) of soil. To determine the role of colonization of M. incognita egg masses by PL251 for biocontrol efficacy, a real-time quantitative polymerase chain reaction (PCR) assay with a detection limit of 10 CFU/egg mass was used. Real-time PCR revealed a significant relationship between egg mass colonization by PL251 and the dose of product applied to soil but no correlation was found between fungal density and biocontrol efficacy or nematode inoculum level. These results demonstrate that rhizosphere competence is not the key mode of action for PL251 in controlling M. incognita on tomato.

Molecular characterization of atoxigenic strains for biological control of aflatoxins in Nigeria.

Aflatoxins are highly toxic carcinogens produced by several species in Aspergillus section Flavi. Strains of A. flavus that do not produce aflatoxins, called atoxigenic strains, have been used commercially in North America as tools for limiting aflatoxin contamination. A similar aflatoxin management strategy is being pursued in Nigeria. In the current study, loci across the 68 kb aflatoxin biosynthesis gene cluster were compared among 18 atoxigenic and two aflatoxin-producing vegetative compatibility groups (VCGs) from Nigeria and an atoxigenic VCG used commercially in North America. Five of the atoxigenic VCGs had large deletions (37-65 kb) extending from the teleomeric side of the aflatoxin biosynthesis cluster. In one VCG (AV0222) the deletion extended through the cluster to the adjacent sugar cluster. The remaining twelve atoxigenic VCGs, including the VCG used for aflatoxin management in North America, contained all the aflatoxin pathway genes, but with defects. Two observations support the long-term persistence of atoxigenicity within A. flavus: first, a comparison of pathway genes revealed more changes in atoxigenic than in aflatoxin-producing isolates relative to the aflatoxin-producing strain NRRL 3357; and second, several non-synonymous changes are unique to atoxigenics. Atoxigenic VCG diversity was assessed with phylogenetic analyses. Although some atoxigenics share relatively recent ancestry, several are more closely related to aflatoxin producers than to other atoxigenics. The current study demonstrates VCGs of A. flavus in West Africa with diverse mechanisms of atoxigenicity and potential value in aflatoxin management programmes.

Effect of different potting systems; inoculation time; nematode density and sources of cereal cyst nematode (Heterodera filipjevi) on juvenile penetration into wheat root system.

Investigations were designed to optimize testing systems for screening wheat breeding lines for resistance to Heterodera filipjevi. The effects of: 1) plant potting systems 2) inoculum level and time of inoculation 3) and type of inoculum of H. filipjevi on detection accuracy were examined in growth chamber experiments in Turkey. The rate of nematode penetration in the highly susceptible variety Bezostaya was used as the base measurement of efficacy. The results showed that the highest level of penetration coupled with high level of germination was obtained in plastic tubes (13 cm long x 3 cm in diam.) when compared to both small flower pots (400 cm3) and smaller plastic tubes (10.2 cm long x 0.8 cm in diam.). The highest rate of nematode penetration into wheat root system was obtained by inoculating the seedlings with 1000 J2 per plant. However, inoculation with 200 J2 at sowing or 200 J2 at sowing plus an additional 200 J2 after germination improved percent penetration when compared to inoculation with 600 or 1000 J2/plant at sowing. The test on the optimum form of inoculum showed that inoculating the seedling with J2's gave the highest rate of nematode penetration over inoculum with eggs or cysts. The results of these experiments demonstrated that screening wheat for resistance can be optimized by raising the seedlings in plastic tubes and inoculating them with 400 J2.

Evaluation of atoxigenic isolates of Aspergillus flavus as potential biocontrol agents for aflatoxin in maize.

Aflatoxin contamination resulting from maize infection by Aspergillus flavus is both an economic and a public health concern. Therefore, strategies for controlling aflatoxin contamination in maize are being investigated. The abilities of eleven naturally occurring atoxigenic isolates in Nigeria to reduce aflatoxin contamination in maize were evaluated in grain competition experiments and in field studies during the 2005 and 2006 growing seasons. Treatments consisted of inoculation of either grains in vials or ears at mid-silking stage in field plots, with the toxigenic isolate (La3228) or atoxigenic isolate alone and co-inoculation of each atoxigenic isolate and La3328. Aflatoxin B(1) + B(2) concentrations were significantly (p < 0.05) lower in the co-inoculation treatments compared with the treatment in which the aflatoxin-producing isolate La3228 was inoculated alone. Relative levels of aflatoxin B(1) + B(2) reduction ranged from 70.1% to 99.9%. Among the atoxigenics, two isolates from Lafia, La3279 and La3303, were most effective at reducing aflatoxin B(1) + B(2) concentrations in both laboratory and field trials. These two isolates have potential value as agents for the biocontrol of aflatoxin contamination in maize. Because these isolates are endemic to West Africa, they are both more likely than introduced isolates to be well adapted to West African environments and to meet regulatory concerns over their use throughout that region.

Comparison of Muscodor albus Volatiles with a Biorational Mixture for Control of Seedling Diseases of Sugar Beet and Root-Knot Nematode on Tomato.

A biorational synthetic mixture of organic components mimicking key antimicrobial gases produced by Muscodor albus was equivalent to the use of live M. albus for control of seedling diseases of sugar beet (Beta vulgaris) caused by Pythium ultimum, Rhizoctonia solani AG 2-2, and Aphanomyces cochlioides. The biorational mixture provided better control than the live M. albus formulation for control of root-knot nematode, Meloidogyne incognita, on tomato (Lycopersicon esculentum). The biorational mixture provided control of damping-off equal to a starch-based formulation of the live fungus for all three sugar beet pathogens, and significantly reduced the number of root-knot galls on tomato roots compared with a barley-based formulation. Rate studies with the biorational mixture showed that 2 and 0.75 µl/cm3 of soil were required to provide optimal control of Rhizoctonia and Pythium damping-off of sugar beet, respectively. Five microliters of biorational mixture per milliliter of water was required for 100% mortality in 24 h for Meloidogyne incognita in in vitro studies. In in vivo studies, 1.67 µl of the biorational mixture/cm3 of sand resulted in fewer root-knot galls than a Muscodor albus infested ground barley formulation applied at 5 g/liter of sand.

Suitability of Pueraria phaseoloides, Chromolaena odorata and Tithonia diversifolia as in-situ mulch for nematode management in musa cropping systems.

Mulching with plant organic matter has been shown to reduce nematode population densities in various cropping systems. The level of nematode control is increased when such mulches are incorporated into the soil as organic amendments. Chromolaena odorata, Tithonia diversifolia and Pueraria phaseoloides are common cover crops in West and Central Africa that produce large quantities of nutrient rich biomass. The aim of this study was to determine, if in-situ mulching of C. odorata, T. diversifolia and P. phaseoloides is suitable for nematode control in Musa production. In a pot trial, the susceptibility of these plants to spiral nematodes was investigated. The effects of different quantities of surface mulch on nematode population densities in the soil and in banana roots also were determined. All mulch types and all quantities led to a reduction in nematode population densities in the soil. The strongest nematode reductions were observed in the Pueraria treatments. In treatments containing banana plants mulching improved plant growth compared to the clean-fallowed soil and induced lower root infestation rates. However, nematode soil populations were higher in mulched than in non-mulched banana treatments. Plant parasitic nematodes also were isolated from roots of all three cover crop species and all three plants caused an increase in nematode numbers in the soil. Therefore, the tested cover crops proved unsuitable for nematode control in a system with the highly susceptible bananas. Further examinations are needed to determine whether or not the positive effects of surface mulching on plantain plant growth and root infestation rates also have positive effects on yield in an in-situ mulching system in the presence of nematodes.

Use of Trichoderma harzianum and Trichoderma viride for the biological control of Meloidogyne incognita on tomato.

Trichoderma harzianum and Trichoderma viride were tested for their capacity to reduce the incidence of the root-knot nematode Meloidogyne incognita on tomato. In vitro studies demonstrated that all tested isolates were effective in causing nematode mortality compared with the control. Trichoderma slightly reduced nematode damage to tomato in vivo. Treatment of the soil with the biocontrol agents before transplanting, improved control over treatment directly at transplanting. The Trichoderma isolates could not be re-isolated from the endorhiza, but were successfully re-isolated from the rhizosphere 45 days after fungal inoculation. Only slight increases in plant growth could be measured. The mutualistic endophyte F. oxysporum 162, used as positive control, was more effective in root-knot nematode biocontrol than the Trichoderma isolates.

Use of high resolution digital thermography to detect Heterodera schachtii infestation in sugar beets.

Thermography is a non-destructive method used to monitor pest and disease infestations, as it is related to changes in plant water status. Surface temperature differences of the crop canopy may be an indicator of nematode infestation as the parasitation of the root system reduces evaporation of leaves. To test the potential of high resolution digital thermography to detect Heterodera schachtii infestation, experiments using increasing nematode densities and different sugar beet varieties were conducted. From June to August 2003 the crop canopy temperature was measured with a thermal infrared camera from a helicopter. A significant correlation between canopy temperature and nematode density was observed with the susceptible cultivar Monza whereas the resistant cultivar Paulina did not show any correlation. Mean temperature comparison showed significant differences between the lowest infestation level (500 eggs and larvae/100 ml soil) and the highest infestation level (>1500 eggs and larvae/100 ml soil). At the beginning of the season canopy temperature differences between healthy and nematode infested sugar beets were higher (approximately 1 degree C) compared to later assessment dates when the water supply in the soil was limited. Since low and high nematode infestation could be clearly distinguished with the susceptible cultivar by airborne thermal images, thermography might be a useful tool for monitoring sugar beet fields.

Efficacy of Paecilomyces lilacinus (strain 251) for the control of Radopholus similis in banana.

Paecilomyces lilacinus is a common soil fungus that has been isolated from many different habitats around the world. It is well known as a facultative egg pathogen of sedentary nematodes and also an important option to control Radopholus similis juvenile and adults in banana. This nematode antagonistic fungus may be used in an integrated approach to control banana plant parasitic nematodes. Dose response and form of application experiments were conducted with burrowing nematode, R. similis, on banana using a commercial water dispersible granulate formulated P. lilacinus (strain 251) product. The results revealed that nematode activity decreased in the presence of this fungus. An important correlation between rates of application and the degree of control of R. simnilis penetration and banana root weight was observed. The best control was achieved in the treatment were plantlets and soil were pre-inoculated with P. lilacinus and reinoculated during transplantation. The results showed that the biocontrol agent P. lilacinus is an excellent candidate for an IPM program against nematodes such as Radopholus similis.

Optimizing the efficacy of Paecilomyces lilacinus (strain 251) for the control of root-knot nematodes.

The egg pathogenic fungus Paecilomyces lilacinus (strain 251) is a biocontrol fungus with a potential range of activity to control the worldwide most important plant parasitic nematodes. This biological nematicide may be an useful tool in an integrated approach to control mainly sedentary nematodes. Greenhouse experiments were conducted with the root-knot nematodes Meloidogyne incognita and M. hapla on tomato. P. lilacinus, formulated as WG (BIOACT WG), was incorporated into soil inoculated with root-knot nematode eggs prior to transplanting the susceptible tomato cultivar "Hellfrucht". Furthermore, soil treatments were combined with seedling treatments 24 hours before transplanting and a soil drench 2 weeks after planting, respectively. Seedling and post planting treatment was also combined with a soil treatment at planting. All single or combination treatments tested decreased the gall index and the number of egg masses compared to the untreated control 12 weeks after planting. However, the combination of the seedling treatment with a pre- or at-planting application of P. lilacinus was necessary to achieve higher levels of control. Additional post plant drenching resulted in only a slight increase In efficacy. To the feasibility of this modified application system for the control of root-knot nematodes, a yield experiment was conducted with M. hapla and the susceptible cultivar "Gnom F1 Hybrid". It could be demonstrated that the above mentioned combination of pre-planting application plus the seedling and one post plant drench gave the best control and resulted in a significant fruit yield increase in concurrence with a decrease in number of galls per root.

Effects of endophytic Fusarium oxysporum towards Radopholus similis activity in absence of banana.

Four endophytic fungi (Fusarium spp.) isolated from the cortical tissue of surface-sterilised banana as well as from tomato roots were tested for their capacity of biological control towards the burrowing nematode Radopholus similis on banana. The pathogenic and parasitic capacities of endophytic fungi towards R. similis were tested in in vitro experiments. No parasitism of fungi on R. similis was observed. However, nematode activity decreased significantly in the presence of all endophytic fungi in vitro when compared to nematodes in the absence of fungi. The effects of fungi on R. similis activities in the soil were tested in the absence of plants. Nematode activities were reduced significantly by 16-30% by endophytic fungi when compared to untreated soil.

Efficacy of Paecilomyces lilacinus (strain 251) for the control of root-knot nematodes.

The egg pathogenic fungus Paecilomyces lilacinus (strain 251), is a unique strain with a wide range of activity against the most important plant parasitic nematodes. Due to increased production capacity by solid state fermentation and a new water dispersible granule (WDG) formulation, this biological nematicide may be used in an integrated approach to control plant parasitic nematodes. Dose response experiments were conducted with the root-knot nematode Meloidogyne incognita on tomatoes using the new WDG formulation. The results revealed a clear correlation between rate applied and the degree of control concerning the reduction in damage to the root and multiplication of the nematode. Best control was achieved by applying the biological nematicide at rates of 2 to 4 times 10(9) conidia per plant as a soil treatment one week before planting. Monitoring the P. lilacinus population in the rhizosphere showed a decline after 2 to 3 month which can lead to insufficient control over a full growing season. Repeated application to maintain the antagonist population at a sufficient level could be used to secure long term control of root-knot nematodes.

Managing the mycorrhizosphere--an approach to sustainable agriculture after the phaseout of methyl bromide?

Plant Health Promoting Rhizobacteria (PHPR) with known biocontrol activity toward the root knot nematode, Meloidogyne incognito, were tested for their ability to promote root colonization of arbuscular mycorrhizal fungi on tomato under greenhouse conditions. In addition, bacteria isolated from mycorrhizal spores were tested for their direct biological control activity on nematode early root penetration. The nematode antagonistic rhizobacteria Rhizobium etli G12 significantly increased mycorrhizal establishment. In addition, two of nine bacteria isolated from the mycorrhizosphere were shown to significantly reduce M. incognita early root penetration. The experiments produced important data on the interaction between rhizobacteria and the symbiotic fungus that in some combinations work synergistically in controlling the root-knot nematode. The potential of using such multitrophic treatment combinations in integrated management targeted first at reducing nematode early root penetration in young transplants and then later for sustainable biocontrol of root-knot under field conditions are discussed.

Risk assessment of biocontrol products: a semi-selective medium for improving quantitative isolation of Paecilomyces lilacinus (strain 251) from soil.

Paecilomyces lilacinus (Thom) Samson 1974 is an egg pathogenic fungus, attacking mainly sedentary stages of root-knot and cyst nematodes, especially eggs, and is about to register as a product for the biological control of nematodes. Monitoring P. lilacinus (strain 251) is essential by way of ascertaining the fate and behaviour of this fungus in the soil. In order to obtain more data on the persistence of P. lilacinus (strain 251) in the soil, an improved selective medium for this strain is necessary. For this reason, different compounds with known antifungal properties were tested. Among them, P. lilacinus (strain 251) was found to tolerate high concentrations of 2,6-dichlor-4-nitroanilin, the active ingredient of Dichloran, which inhibits growth of many commonly occurring soil-borne fungi. The fact that P. lilacinus is more tolerant of higher salt concentrations than many other fungi are, was also taken into consideration. As a basis for the improved medium, OHIO-agar medium was used.

Evaluation of Trichoderma spp. for biocontrol of tomato sudden caused by Pythium aphanidermatum following flooding in tropical hot season.

Tomato sudden death is a major problem in tomato production in tropical lowland areas. The plant wilts and dies following artificial or natural flooding for 48-72 hrs in the summer season. Occurrence of this disease is related to aggressiveness of Pythium aphanidermatum on tomato at high soil temperature (>30 degrees C). Several methods such as using biological control agents, fungicides and other cultural practices were applied in attempts to control tomato sudden death. Three Trichoderma harzianum and two Trichoderma virens isolates were evaluated for biocontrol of the disease in the greenhouse and in the field T. harzianum and T. virens isolates were separately used to treat the seed, potting medium and also incorporated into the soil before transplanting. Field soil was naturally infested with P. aphanidermatum, while greenhouse soil was inoculated with the pathogen 10 days after transplanting. All treatments were flooded for 48 hrs at 32 degrees C soil temperature. Results from this study show that most tested T. harzianum and T. virens isolates have little promise for control of tomato sudden death following flooding. The percentage of tomato plants that wilted after growing in soil treated with either T. harzianum or T. virens and P. aphanidermatum was not significantly different when compared to the soil treated with P. aphanidermatum alone. No wilted plants were observed in the control (non treated soil).

Effect of seed treatment with organic acids on the control of common bunt (Tilletia tritici and T. laevis) in wheat.

Common bunt caused by Tilletia tritici and T. laevis is an important disease, causes considerable losses in wheat yield on a world wild. The disease reduces yields, complicates harvesting and lowers the quality of the grain. It occurs more frequently and causes greater damage on winter wheat than on spring wheat. Grain standards designate wheat that has an unmistakable odor of smut or that contains smut balls, portions of balls, or spores of smut in excess of a quantity equal to 14 balls of average size in 250 g of wheat as "light smutty". Samples containing an excess of 30 balls, or their equivalent, in 250 g of wheat are graded as "smutty". Grain in these grades brings lower prices. Using commercial acetic acid and lactic acid, the pathogen was successfully controlled, but the treatment negatively affected seed germination and seedling vitality. Using dilutions of acetic acid and lactic acid, significant control of the pathogen also was achieved with acetic acid without causing phytotoxicity. Dilutions of lactic acid also gave good control, but showed some phytotoxicity. Using 30-50 ml/kg of vinegar, which is a natural source of acetic acid, proved to be one of the most effective alternatives for control of common bunt on wheat. The treatment had no negative effects on seed germination nor on seedling vitality.

Effects of natural products on soil organisms and plant health enhancement.

TerraPy, Magic Wet and Chitosan are soil and plant revitalizers based on natural renewable raw materials. These products stimulate microbial activity in the soil and promote plant growth. Their importance to practical agriculture can be seen in their ability to improve soil health, especially where intensive cultivation has shifted the biological balance in the soil ecosystem to high numbers of plant pathogens. The objective of this study was to investigate the plant beneficial capacities of TerraPy, Magic Wet and Chitosan and to evaluate their effect on bacterial and nematode communities in soils. Tomato seedlings (Lycopersicum esculentum cv. Hellfrucht Frühstamm) were planted into pots containing a sand/soil mixture (1:1, v/v) and were treated with TerraPy, Magic Wet and Chitosan at 200 kg/ha. At 0, 1, 3, 7 and 14 days after inoculation the following soil parameters were evaluated: soil pH, bacterial and fungal population density (cfu/g soil), total number of saprophytic and plant-parasitic nematodes. At the final sampling date tomato shoot and root fresh weight as well as Meloidogyne infestation was recorded. Plant growth was lowest and nematode infestation was highest in the control. Soil bacterial population densities increased within 24 hours after treatment between 4-fold (Magic Wet) and 19-fold (Chitosan). Bacterial richness and diversity were not significantly altered. Dominant bacterial genera were Acinetobacter (41%) and Pseudomonas (22%) for TerraPy, Pseudomonas (30%) and Acinetobacter (13%) for Magic Wet, Acinetobacter (8.9%) and Pseuodomonas (81%) for Chitosan and Bacillus (42%) and Pseudomonas (32%) for the control. Increased microbial activity also was associated with higher numbers of saprophytic nematodes. The results demonstrated the positive effects of natural products in stimulating soil microbial activity and thereby the antagonistic potential in soils leading to a reduction in nematode infestation and improved plant growth.