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 Pasteuria penetrans (Thorne) Sayre & Starr has been recognized as one of the most promising biological agents for the control of root-knot nematodes (Sayre and Starr., 1989; Stirling., 1984). P. penetrans is widely distributed throughout the world and contributes natural control of nematodes, especially of root-knot nematodes (Chen and Dickson, 1998). P. penetrans is gram-positive bacterium and has various characteristics desirable for a biological control agent against plant-parasitic nematodes (Dickson and Oostendorp, 1990). The endospores can survive under adverse environmental conditions such as humidity, desiccation, high and low temperature, and absence of host nematodes for a long period (Dickson et al., 1994). Storage of P. penetrans for 11 years did not reduce the ability of the endospores to attach m to juveniles of M. javanica (Giannakou et al., 1997).

 P. penetrans could reduce yield losses caused by M. incognita 23-24% for tobacco and 38-55% for winter vetch in two years experiments (Brown et al., 1985). In Korea, occurrence of P. penetrans was reported from various plant-parasitic nematode species of Meloidogyne spp., Helicotylenchus sp., Pratylenchus sp., Heterodera sp., and Aphelenchus sp. (Cho et al., 2005a). The Korean isolates of P. penetrans also showed promising control effects on major root-knot nematodes (Yu et al., 2003; Zhu et al., 2005; Park et al., 2005). P. penetrans 98-35 isolate showed control effect of 92% on M. arenaria in a pot test (Cho et al., 2000).

 The characteristic of P. penetrans as an obligate nematode parasite is a major limiting factor in using the bacterium for biological control of nematodes (Williams et al., 1989; Cho et al., 2005b). Artificially cultured P. penetrans was already commercialized, however, the product was not successful in effectively managing Belonolaimus longicaudatus on golf course turf (Crow et al., 2011).

 For mass production of P. penetrans, it is important to understand the endospore attachment characteristics to the juveniles of root-knot nematodes (Freitas et al., 1997). The endospores attach to the cuticle of a nematode and a penetration tube develops which penetrates the nematode. Host adhesion of the endospore is one of the most critical steps in completion of the life-cycle (Chen and Dickson, 1998).

 This study was conducted to understand the effects of temperatures and root-knot nematode species on attachment of the endospores to root-knot nematode juveniles.

Materials and Methods

 Pasteuria penetrans infested soil was collected at an oriental melon (Cucumis melo var. Makuwa, Makino) greenhouse in Seongju-county, Kyungsangbook-do, Korea in 1998. The plant debris and large particles in the soil were removed by sieving through 3 mm-diameter sieve and stored in a plastic bucket and covered with plastic film in room temperature before it was used for the experiments.

Meloidogyne arenaria (Neal) Chitwood was collected from roots of oriental melon (Cucumis melo var. Makuwa, Makino) in Seongju-county, Kyungsangbook-do and maintained on tomato (Lycopersicon esculentum Mill. cv. Young-gwang) roots in greenhouse at National Institute of Horticultural & Herbal Science, Suwon, Korea. The juveniles (J2s) were collected from the tomato roots by combining Hussey and Barker's method and Baermann funnel method. The J2s not older than three days after hatching were used in the experiment.

Effects of temperature on P. penetrans endospore attachment to M. arenaria juveniles

 The soil was put in 87×15 mm plastic petri dishes. Each petri dish containing 50g soil was inoculated with 3,000 J2 in 15cc distilled water. The inoculated petri dishes were sealed with parafilm. The dishes were put in incubators set at different temperatures of 20℃, 25℃, 30℃, and 35℃. Each treatment was replicated 3 times.

 After 7 days, the J2s were recovered from the petri dishes by combined sieving and centrifugal sugar flotation method. The recovered J2s were examined under inverted microscope (Zeiss, Axiovert 135) to check the endospore attachment and count the endospore numbers per J2 on 20 individuals from each dish.

 To check the presence of live J2s in the test soil, 3 petri dishes inoculated with 15cc distilled water were incubated in 25℃. From the soil inoculated with distilled water only, no J2 was recovered. Statistical analysis was done using SAS program.

Effects of P. penetrans infested soil pre-exposure temperatures on the endospore attachment to M. arenaria juveniles

 The P. penetrans infested soil used in this test was the same soil as described above. The soil was stored in a plastic container under room temperature for 15 months until this experiment.

 The plastic petri dishes containing the 50g soil and sealed with parafilm were incubated for 10 days in different temperatures of -30℃, 4℃, 40℃, 50℃ and room temperature. Glass petri dishes containing 50g soil and sealed with aluminum foil were incubated in 100℃ dry oven for 8 hours. After incubations in the different temperature conditions, the petri dishes were inoculated with 3,000 J2s each as described above. The dishes were incubated in 25℃ for 3 days. The J2s were recovered and examined as described above.

P. penetrans endospore attachment to 3 Meloidogyne species juveniles

 The soil infested with P. penetrans was tested for endospore attachment to juveniles of different Meloidogyne species. M. incognita was collected from roots of oriental melon in Seongju and maintained on tomato (Lycopersicon esculentum Mill. cv. Young-gwang). M. hapla, collected from peony (Paeonia lactiflora Pall.) root at Euisung, Korea, was maintained on tomato (L. esculentum Mill. cv. Seo-gwang). The soil infested with P. penetrans was prepared and the J2s of the three species including M. arenaria were inoculated as described above. Attachment rates and endospore numbers were examined after incubation at 25℃ for 3 days.

Results and Discussions

Effects of temperature on P. penetrans endospore attachment to M. arenaria juveniles

 Attachment rates and numbers of Pasteuria penetrans endospores to Meloidogyne arenaria juvenile (J2) showed significant difference affected by the incubation temperatures. Among the four incubation temperatures tested, the endospore numbers attached per J2 were highest at 25℃ with 28.3/J2 followed by 30℃, 20℃, and 35℃ (Table 1). In the all temperatures, all the J2s were encumbered with at least one endospore.

Table 1. Pasteuria penetrans endospore attachment to Meloidogyne arenaria juveniles (J2) affected by incubation temperatures^*

 Hatz and Dickson (1992) reported that the optimum temperature for attachment was 30℃ when M. arenaria and P. penetrans infested soil were incubated for 24 hours at 10℃, 20℃, 30℃, and 35℃. In the report, incubation in temperature of 25℃ was not included. In our results, the optimum temperature for the endospore attachment was 25℃, and this result confirms the regression model of Freitas et al., (1997) presenting the maximum attachment would occur when J2 and the endospores were incubated at approximately 25℃.

Effects of P. penetrans infested soil pre-exposure

 The Pasteuria penetrans infested soil pre-exposure to above lethal temperatures significantly lowered the endospore attachment (Table 2). Soil pre-exposure to high temperatures of 40℃ and 50℃ significantly reduced the endospore attachment numbers up to 1/J2 which was much lower than 5.3/J2 of room temperature. Attachment rates also decreased to 8.3 and 5.0% in 40℃ and 50℃, respectively, while that of room temperature was 60%. After the soil pre-exposure to 100℃ for 8 hours, endospore attachment did not occur. In contrast to the high temperature exposure, effects of the soil pre-exposure to lower temperatures on the endospore attachment were not that significant.

Table 2. Pasteuria penetrans endospore attachment to Meloidogyne arenaria juveniles inoculated to the bacterium infested soil pre-exposed at different temperatures

 The attachment rates were 25 and 31.7% after soil pre-exposure at -30℃ and 4℃, respectively, while that of room temperature was 60% (Table 2). The endospore numbers per J2 also showed slight decrease to 3.5 and 4.3/J2 while that of room temperature was 5.3/J2. Freitas et al. (1997) also observed the decreased endospore attachment rate after endospore encumbered soil pre-exposure at 50℃ and higher. Dutky and Sayre (1978) observed that there was no endospore attachment after P. penetrans infested soil exposed to 130℃ for 1 hour. Giannakou et al. (1997) showed that preheating P. penetrans spores to above normal temperatures (60°C) significantly increased attachment but reduced infection to M. javanica J2s. These results showed that higher temperatures were more fatal to the vitality of P. penetrans endospores than lower temperatures. And our results showed that the P. penetrans in the infested soil stored for 15 months under room temperature still maintained its ability to attach to root-knot nematode J2s although the endospore attachment rates and numbers were lower than those of the first experiment (Table 1 and 2).

P. penetrans endospore attachment to 3 Meloidogyne species juveniles

 Attachment rates and numbers of Pasteuria penetrans endospores to Meloidogyne arenaria juvenile were 100% and 56.7/J2. However, there was no endospore attached to the J2s of M. hapla and M. incognita (Table 3). Different endospore attachment among P. penetrans isolates on Meloidogyne spp. and Pratylenchus sp. (Oostendorp et al., 1990) and variation in host ranges of P. penetrans from California, U.S.A. and Australia were demonstrated (Stirling, 1985). The endospores of U.S.A. isolate were readily attached to M. javanica, M. incognita and M. hapla, but three P. penetrans populations from Australia were more specialized. Our result indicates that the P. penetrans used in this experiment has very high host specification with M. arenaria.

Table 3. Pasteuria penetrans endospore attachment to three Meloidogyne species juveniles after incubation for 3 days at 25℃

 The above experiment results showed that the P. penetrans endospore attachment to root-knot nematode juvenile is more feasible in 25℃ condition than in other temperatures. And the soil pre-treatment in lower and higher than room temperature greatly reduced the attachment of the endospores to the root-knot nematode juveniles. And the P. penetrans showed its host specific endospore attachment only on M. arenaria. This information on the temperature effects on endospore attachments and the host specificity of P. penetrans used in this study would be useful for a development of mass production system of P. penetrans and practical use of the bacterium for biological control of root-knot nematodes in the future.

Acknowledgement

 This study was partly supported by research fund (PJ0083412013) from Rural Development Administration, Republic of Korea.