Biological control of fungal and bacterial plant pathogens IOBC/wprs Bulletin Vol. 43, 2009 pp. 297-299
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Understanding multitrophic interactions to facilitate successful biocontrol of plant-parasitic nematodes with Paecilomyces lilacinus strain 251
Sebastian Kiewnick
Agroscope Changins-Wädenswil, Research Station ACW, Zoology/Nematology, Schloss P.O.Box 185, 8820 Wädenswil, Switzerland, E-mail: sebastian.kiewnick@acw.admin.ch
Abstract:The facultative egg-pathogenic fungus Paecilomyces lilacinus strain 251 (PL251) is one of the most widely tested biocontrol agents for control of plant-parasitic nematodes. Recently, PL251 was included as active substance in Annex I to the directive 91/414/EEC. In the USA, PL251 is registered as bio-nematicide under the trade name MELOCON®WG for use on a variety of crops. So far PL251 has demonstrated efficacy in reducing root-knot, cyst and free living plant-parasitic nematodes on a range of crops. However, to better understand the multitrophic interactions of PL251 with host- or non-host plants, nematodes, mutualistic fungal endophytes, and mycorrhiza studies were conducted to determine their importance for biological efficacy. In none of the studies conducted, adverse effects on mutualistic fungal endophytes, mycorhiza, fungal antagonists or entomopathogenic nematodes were observed. Conversely to other nematophagous fungi, rhizosphere competence seems not a key factor for the efficacy of PL251. However, studies are underway to determine the eggmass colonisation by PL251 using realtime PCR assays which are able to detect 10 CFU per eggmass or less. Monitoring the persistence of PL251 under field conditions using dilution plating techniques and nested PCR revealed a rapid decline of the fungal density in soil over time. Although detection of PL251 in soil was still possible two years after application, the overall suppressiveness of egg pathogenic fungi towards cyst nematodes was not affected.
Key words: biological control, root-knot nematode, BIOACT WG
Introduction
Paecilomyces lilacinus is a facultative pathogen of eggs of root-knot and cyst nematodes. P.
lilacinus strain 251 (PL251) is commercially formulated and registered as a biological nematicide under the product names BIOACT®WG and MELOCON®WG in Europe and USA, respectively. Following application to the soil, PL251 reduces the nematode population by infecting mainly eggs and to a lesser extend juveniles and sedentary stages. The efficacy of PL251 has been shown in multiple greenhouse and field experiments, a pre-requisite for successful commercialization of a biocontrol product (Kiewnick & Sikora, 2006a; 2006b;
Kiewnick, 2007a). However, still many questions remain unanswered concerning the factors and multitrophic interactions that affect the biocontrol potential of PL251.
Understanding the dynamics of a biological control agent (BCA) is important for predicting the success of biocontrol (Bidochka, 2001). Many BCAs require a certain population density to control pests or diseases, therefore persistance and survival of the antagonist for a certain period is important. As the impact of multitrophic interactions on antagonistic fungi should be taken into consideration when biocontrol is employed, ecological studies can help to facilitate efficacy of biocontrol agents. Molecular tools can help in understanding the factors important for efficient biocontrol of nematodes using PL251.
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Therefore, a range of attempts are presented to understand the role of multitrophic interactions in the biocontrol of plant-parasitic nematodes by PL251.
Material and methods
Persistence in the field
The potential of PL251 to persist and establish in the environment was evaluated in field experiments conducted in a sugar beet field using a broadcast application of the commercial product BIOACT® WG. The fungal antagonist was applied at a rate of 4 and 12 kg product per ha in 2003 and 2004, respectively, and incorporated into the soil prior to planting sugar beets. The population dynamics of PL251 was monitored using dilution plating techniques.
As a more sensitive method of detection, regular PCR plus nested PCR was used for monitoring the long term establishment of PL251 in the field.
Interaction between fungal antagonist and nematode
To investigate the interaction between PL251 and the root-knot nematode M. incognita, the quantification of the fungus on the egg masses was done by real-time PCR according to the protocol developed by Atkins et al. (2005). They used TaqManPCR primers and probes, which were designed from the ITS region of the fungal genome and had been confirmed as being species specific. The fluorogenic probe (PLrtP) was labelled at the 5’ end with the fluorogenic reporter dye FAM (6-carboxy-fluorescein), and the 3’end was modified with the quencher dye TAMRA (6-carboxy-tetramethylrhodamine). To generate a standard curve for real-time PCR quantification, 10 egg masses were taken from roots of a tomato plant infected with M. incognita and inoculated with 107 viable conidia of PL251. Whole DNA from egg masses was extracted with a plant DNA extraction kit (Mo-Bio). Serial dilutions were prepared from each sample so that 3 µl represented 1 × 105, 1 × 104 to a final dilution of 10 CFU per egg mass. DNA prepared from the non-inoculated M. incognita egg masses was used as a negative control. Real-time PCR was performed in Applied Biosystems fast optical 96- well 0.1 ml reaction plates covered with AB optical adhesive covers with the automated ABI Prism 7500 sequence detector (Applied Biosystems). Primers PLrtF and PLrtR were included at a final concentration of 900 nM each and the TaqManprobe (PLrtP) was used at 225 nM.
The thermal cycle protocol for PCR amplification was used according to Atkins et al. (2005) and reactions were performed in triplicate.
Results and discussion
Persistence in the field
Using soil dilution plating techniques, it was demonstrated that in both years the initial density of the fungal antagonist after application was significantly lower than predicted and the spatial distribution was very heterogeneous. In 2003, the density of PL251 had already decreased by more than 90% at 90 days after application, and at 120 days the fungus was no longer detected in soil. In 2004, PL251 was detected in low numbers at sugar beet harvest 200 days past application. Correlation analysis showed that the exponential decline rate of PL251 in field soil was independent from the initial spatial distribution and not altered by the population dynamics of the sugar beet cyst nematode H. schachtii. Furthermore, the high level of parasitism of H. schachtii eggs by indigenous soil fungi already present in this soil was not altered by the application of PL251, which demonstrated the low level of establishment of PL251 in a soil that has been known to be suppressive to H. schachtii (Pyrovolakis et al., 1999).
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Further monitoring of PL251 in these fields in spring 2005 using regular as well as nested PCR showed that the biocontrol fungus could be detected in all treated but also the untreated plots (Kiewnick, 2007b). However, there were no significant differences in fungal density between treated and untreated plots and the density of PL251 was far below the background level of other filamentous fungi demonstrating the lack of establishment under field conditions.
Interaction between PL251 and Meloidogyne incognita
Based on the data obtained from the real-time PCR quantification assays, a standard curve was generated by using data derived from the serial dilution of PL251 from egg masses. With this method, it was possible to detect 10 CFU of PL251 per root-knot nematode egg mass or less. Therefore, real-time PCR can be used to monitor the density of PL251 on or in egg masses to help in understanding the multitrophic interactions between the biocontrol fungus, the nematode and the host plant. However, preliminary data from the experiments on the interaction of antagonist dose and nematode inoculum density indicate no correlation between biocontrol efficacy and egg mass colonization. Future research will therefore focus more on the interaction between PL251 and the target nematode, and to further identify the factors important for successful biological control of nematodes.
References
Atkins, S.D., Clark, I.M., Pande, S., Hirsch, P.R. & Kerry, B.R. 2005: The use of real-time PCR and species-specific primers for the identification and monitoring of Paecilomyces lilacinus. – FEMS Microbiol. Ecol. 51: 257-264.
Bidochka, M.J. 2001: Monitoring the fate of biocontrol fungi. In: Butt, T.M., Jackson, C.W.
& Magan, N. (eds.) Fungi as Biocontrol Agents: Progress, Problems and Potential. – CABI Publishing, Oxon, UK.
Kiewnick, S. 2007a: Practicalities of developing and registering microbial biological control agents. – CAB Rev. 2: 1-11.
Kiewnick, S. 2007b: Importance of multitrophic interactions for the efficacy of Paecilomyces lilacinus strain 251 to control root-knot nematodes. – J. Nematol. 39: 72.
Kiewnick, S. & Sikora, R.A. 2006a: Biological control of the root-knot nematode Meloido- gyne incognita by Paecilomyces lilacinus strain 251. – Biol. Contr. 38: 179-187.
Kiewnick, S. & Sikora, R.A. 2006b: Evaluation of Paecilomyces lilacinus strain 251 for the biological control of the northern root-knot nematode Meloidogyne hapla Chitwood. – Nematology 8: 69-78.
Pyrowolakis, A., Schuster, R.P. & Sikora, R.A. 1999: Effect of cropping pattern and green manure on the antagonistic potential and the diversity of egg pathogenic fungi of the beet cyst nematode Heterodera schachtii. – Nematology 1: 165-171.
