In Vitro Cultivation of Aphelenchoides besseyi Christie on Fungal Cultures

Aphelenchoides is a genus that is consisted of parasitic nematodes on high-level plants, associated with insects, and most species are microphagous nematodes. Some Aphelenchoides species recognized as major pests include Aphelenchoides besseyi Christie, Ap. ritzemabosi (Schwartz) Steiner and Buhrer, Ap. fragariae (Ritzema Bos) Christie and Ap. arachidis Bos. (De Waele, 2002). Ap. besseyi are recognized to cause white tips on rice and are distributed in rice fields around the globe (EPPO, 2017).

Ap. besseyi are able to reproduce parthenogenically or amphimictically. Parthenogenesis is an asexual reproduction where female nematodes produce eggs cells without fertilization. This behavior causes nematodes to be able to produce many offspring. However, competition to obtain nutrient also increases. Amphimictic is a sexual reproduction where sperm and egg cells from different individuals meet (cross fertilization). Nematodes are able to mate freely and produce fertile offspring. Population of Ap. besseyi from Russia were able to parthenogenesis on Fusarium solani cultures (Sudakova & Stoyakov, 1967), while Ap. besseyi populations from Taiwan were able to reproduce amphimictically on Aerobasidium pullulans cultures (Huang et al., 1979). To our knowledge, until today there has not been scientific information on mass rearing of Ap. besseyi populations from Indonesia on fungal culture mediums. Mass rearing techniques for Ap.besseyi is essential for further research. Therefore, this study aims to investigate fungal species medium and temperature combinations for optimum Ap. besseyi rearing.

Ap. besseyi and Fungal Culture
Ap. besseyi used in the study were collected from rice seeds, variety Pak Tiwi-1, and obtained from Balai Besar Padi Sukamandi Subang. Nematode extraction was done using the Baermann funnel method based on procedures from International Seed Testing Association (ISTA) (2018), by cutting 5 g of seed hilum. After cutting hilum, seeds were placed on cloths and immersed in water. Incubation was done for 24 hours at 25°C. Incubation results were extracted using a 500 mesh. Ap. besseyi surfaces were sterilized using streptomycin sulphate (0.l%) for 10 minutes followed by rinsing wth sterilized water for 3 times. Fungal species used as rearing medium of Ap. besseyi were Alternaria padwickii, Fusarium semitectum, and Botrytis cinerea. Cultures were obtained from the Mycology Laboratory, Faculty of Agriculture, Bogor Agriculture Institute. Fungal colonies were grown on Potato Dextrose Agar (PDA) medium on 9-cm petri dishes. Fungal cultures were incubated at 25 o C and grown until entire surfaces of medium were covered with mycelium.

Ap. besseyi rearing on Fungal Cultures
Twenty five nematodes were infested to each petri dish when mycelium of tested fungal species has entirely covered the medium. Each petri dish was stored in a black plastic bags and incubated in dark conditions at temperatures of 20ºC, 25ºC, and 30ºC. Nematodes were harvested 21 days after infestation, followed by population counts. Mediums were sliced and extracted using a modified Baermann funnel method. Numbers of juvenile and mature nematodes were calculated using the formula from

Experimental Design and Data Analysis
This study was designed as a Factorial Complete Randomized Design. The first factor were the three fungal species used as medium and the second factors were the three incubation temperatures Treatment combination tested were nine and each treatment combination consisted of nine replications. Data obtained were processed using Excel 2013 and analyzed using Minitab 16 Statistical Software with a Tukey post-hoc test at 95% confidence level.

RESULTS AND DISCUSSION
Fungal species used as rearing medium of Ap. besseyi are all considered as plant pathogens. Al. padwickii and F. semitectum are pathogen that infect rice seeds in the fields and storages, while B. cinerea is a pathogen on strawberries which causes grey mold. Colony of Al. padwickii on PDA medium were gray with smooth mycelium. Colony located on the underside of the PDA medium was black. Conidiophores were dark colored, elongated, and conidia contained 7-8 septas that were oval shaped. Macromorphological characteristics of F. semitectum on PDA mediums contained dense mycelium and was white to brownish depending on its age. Abundant microconidia were oval shaped, in general contained three septas, hypha microscopically were transparent and possessed septas. Colonies of B. cinerea on PDA medium were greyish brown with white cotton-like mycelium on the edges. Conidiophores were long, slim, contained hyaline, irregularly branched on top, and apical cells were larger and round (Figure 1). Rearing of Ap. besseyi resulted in various growth stages of nematodes, including juvenile to mature nematodes ( Figure 2). Female nematodes were elongated, with lengths of 0.66-0.75 mm and slender. Mouths were round, did not contained striations and in general were wider than their neck or parallel with their body width. Heads were flat and round on its anterior with a strong and sclerotized supporting structure, which forms the head structure and function to point their thin and slightly offset stylets. Cuticles were smooth annulation. Sizes and shapes of male nematodes were similar to females, except for the characteristics of their reproduction structure.
Results of Ap. besseyi rearing on three culture species showed that nematode population varied between culture species and incubation temperature. Population of Ap. besseyi reached the highest number, 9,115 individuals, when reared on Al. padwickii at 25°C, while the lowest population were obtained from nematodes reared on B. cinerea at 30°C (Table 1). Optimum temperature for Ap. besseyi development is 21-25°C, their life cycles last for about 10 days at 21°C, eight days at 23°C, and nematode did not grow at 13°C (Bridge et al., 2005).
On fungal mediums, Ap. besseyi have been reported to be able to finish their life cycle at 20°C (Rajan et al., 1990) and between 23-30 °C (Huang et al., 1972  the effects of temperature on embryo development is one the main factor that affect nematode ecology and distribution. Reproduction factor of Ap. besseyi was the highest on Al. padwickii and F. semitectum incubated at 25°C and Al. padwickii at 20°C. Reproduction factor of Ap. besseyi reared on Al. padwickii incubated at 25°C reached 364.6 times implying that Ap. besseyi were able to complete their life cycle and reproduce. However, when reared on B. cinerea at 25°C, reproduction factor only reached 5.5 times. Reproduction factor of nematodes reared on B. cinerea at 25°C was lower compared to nematodes reared on F. semitectum incubated at 30°C, specifically 6.3 times. This implies that Ap. besseyi were still able to survive and reproduce on F. semitectum at 30°C even though conditions were not optimum (Table 1)  Food source is an important factor that determines the increase of nematode populations. Food preferences of Ap. besseyi can be evaluated based on the population of mature nematodes from different fungi cultures. Several fungal species have essential function and can be considered to increase Ap. besseyi reproduction in vitro (Rajan et al., 1990;Jamali et al., 2008). The highest Ap. besseyi population was obtained from Al. padwickii grown on PDA mediums incubated at 25ºC. PDA was the best substrate for growing fungi used for Ap. besseyi rearing. Nematodes prefer mycelium rather than conidia as food source. Therefore, nematodes are not able to reproduce and survive on medium that induct fungi sporulation (Jamali et al., 2008).
At 25ºC, reproduction factor of nematodes reared on Al. padwickii was 364.6 times, while F. semitectum was 253.4 times. Therefore, Al. padwickii grown on PDA and incubated at 25ºC may be an alternative to rear Ap. besseyi in laboratories for ecological, epidemiological, and molecular testing. However, Al. padwickii grows slower than F. semitectum causing longer time for preparation before being able to be inoculated with Ap. besseyi.

CONCLUSION
All three fungal species were suitable as mass rearing media for Ap. besseyi with population density depending on the combination of fungal species and temperature. Between all fungal species and temperature combinations, Al. padwicki inoculated at 25ºC was the best combination for Ap. besseyi mass rearing.