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.

Barcoding quarantine nematodes and their close relatives: an update on the QBOL-project.

Identification of plant pests, in particular quarantine species, needs to be fast and accurate to enable timely plant protection measures. In addition, a false diagnosis can cause serious financial losses for trade and producers. It is now well established that genetically based diagnosis is a reliable alternative to the classical identification procedures generally based on morphological features, which usually require expert taxonomic skills. On the other hand, genetic diagnosis through the use of DNA-barcodes, i.e. stretches of DNA that contain taxon-specific information, can be performed by any skilled laboratory worker. The European Union 7th framework project QBOL aims to establish DNA-barcodes for all European quarantine organisms as well as their close relatives. The results and protocols will be disseminated in the publicly available and curated database Q-BANK. To enable genetically based identification requires knowledge of the genetic variation both within and between the species of interest as well as their close relatives. For the nematodes, several gene regions (the COI, COII, SSU, LSU and RNA polymerase subunit II) are being evaluated for their barcoding potential.

First Report of Root-Knot Nematode Meloidogyne enterolobii on Tomato and Cucumber in Switzerland.

Severe stunting and extensive root galling were observed on tomato rootstock (Solanum lycopersicum L. cv. Maxifort) resistant to Meloidogyne incognita (Kofoid & White, 1919) Chitwood, 1949, M. javanica (Treub, 1885), and M. arenaria (Neal, 1889) Chitwood, 1949 and cucumber (Cucumis sativus L. cv. Loustik) from two commercial greenhouses in the cantons Aargau and Lucerne in northern Switzerland. Examination of the roots of infected plants revealed the presence of root-knot nematodes in large numbers. Juveniles, males, and females were isolated, and the species was determined on the basis of morphological characteristics, including the female perineal pattern. Identification was confirmed by female esterase (Est) and malate dehydrogenase (MdH) electrophoresis (20 each for Est and MdH). All methods of identification were consistent with M. enterolobii Yang & Eisenback, 1983 (4). For further confirmation, type material of M. enterolobii (from the original host Enterolobium contortisiliquum (Vell.) Morong) from China (4) was used. Furthermore, comparison of the sequence data from 12 individuals of each of the two Swiss populations and the type material of a 310-bp fragment of cytochrome oxidase I (COI), a 723-bp fragment covering the internal transcribed spacer (ITS) region 1, 5.8s, ITS2, and part of the 26s, the mtDNA 63-bp repeat region, and a 780-bp fragment of the intergenic spacer region (1-3) showed 100% homology and confirmed the identification as M. enterolobii. The species M. enterolobii is of great importance because it is able to reproduce on resistant tobacco, pepper, watermelon, and tomato (4). To our knowledge, this is the first report of M. enterolobii in Switzerland. References: (1) M. A. M. Adam et al. Plant Pathol. 56:190, 2007. (2) V. C. Blok et al. Nematology 4:773, 2002. (3) T. C. Vrain et al. Fundam. Appl. Nematol. 15:565, 1992. (4) B. Yang and J. D. Eisenback. J. Nematol. 15:381, 1983.

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.

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.

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.