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Home>FFTC Document Database>Extension Bulletins>Current Status of Bio-Pesticides Development, Farmer's Acceptance and Utilization, and Future Perspective in Taiwan
Current Status of Bio-Pesticides Development, Farmer's Acceptance and Utilization, and Future Perspective in Taiwan
Suey-Sheng Kao
Biopesticides Division
Taiwan Agricultural Chemicals and Toxic Substances Research Institute
Council of Agriculture, Wufeng, Taichung, 413
Taiwan ROC, 2009-03-25


Taiwan has made significant progress in the development and application of bio-pesticides and microbial pesticides in the past two decades. Sex pheromones of Cylas formicarius elegantulua, Eucosma notanthes, Spodoptera litura, and S. exgiua have been synthesized, formulated, and utilized in many ways with satisfactory results, including monitoring, mass trapping, and mating disruption. Nomurea riley was found to be pathogenic to Heliocoverpa armigera and Spodoptera exigua. It effectively controlled H. armigera during field applications. Metarhizium anisopliae was applied to control S. exigua, Brontispa longissima, Loadelphax striatellus. M. anisopliae was also used for destruxin production. Destruxins produced showed high virulence to S. exigua. Beauveria bassiana in soil was found to be lethal to Cylas formicarius. B. bassiana preparations were effective in controlling C. formicarius, Ostrinia furnacalis, and Riptorus lineasis. B. bassiana was also pathogenic to Lipaphis erysimi. The optimal growth and maximum sporulation condition of three isolates of Verticillium lecanii were investigated as well. V. lecanii was reported to be highly pathogenic to Myzus persicae, Macrosiphoniella sanborni, Toxoptera aurantii, Liaphis erysimi, Aphis gossypii, and Saissetia oleae. Many aspects relating to these fungi, e.g., characterization, breeding fungicide-resistant mutants, production process, recovery, formulation, factors affecting infectivity, and compatibility with pesticides, were also evaluated. Granulosis viruses of Plutella xylostella, Artogeia rapae, and nuclear polyhedrosis viruses of Spodoptera litura, and S. exigua were identified and field tested against their own hosts. Heliothis NPV introduced from the United States was effective in controlling H. armigera. Tests were also conducted to evaluate the effectiveness of various adjuvants additive and UV protectants. The host spectra of Autographa californica nucleopolyhedrosis virus were examined. Attempts were also made to genetically improve the activity of A. californica nucleopolyhedrosis virus. Construction of recombinant AcMNPV containing enhanced green fluorescence protein (EGFP) gene or red fluorescence protein (DsRed) gene as tracer for environmental risk assessment was also attempted. On the other hand, Bacillus thuringiensis (Bt) is so far the only microbial insecticide registered for insect control against P. xylostella, A. rapae. etc. Isolation and characterization of local Bt isolates achieved fruitful results and obtained many novel cry genes. Transgenic Bt-bacterium showed a good insecticidal effect against P. xylostella. The efficacy of Bt has been enhanced by adding feeding stimulants, adjuvants and UV protectants, or microencapsulation. Recently, the bioactivity of entomopathogenic- nematophilic bacterium and Photorhabdus luminescen- was evaluated. Results showed that it exhibited high insecticidal and antimicrobial activities against local insect pests and plant pathogens. The plantform technology for mass production of microbial fungicides was established with the use of serial plot-scade liquid fermentors. High yield of durable biomass for targeted microbial groups (Streptomyces spp., Bacillus subtilis, and Gliocladium virens) was achieved. Their effectiveness in disease control was satisfactory. Such positive results from the tested biopesticides pushed this study to recommend further studies on their effective use against agricultural pests.

Key words: bio-pesticides, bio-insecticides, bio-fungicides, entomopathogenic fungi, Bacillus thuringiensis, baculoviruses, Photorhabdus luminescens pheromones


Taiwan is located in tropic and subtropic areas with high temperatures and humidity, prone to infestations by many pest insects. In the 1970s, many evidences indicated that the overuse of chemical insecticides to control insect pests caused many negative side effects. For this reason, during the 1980s and thereafter, the Council of Agriculture (COA) of Taiwan has promoted the development of bio-pesticides. Since then, many active programs on bio-pesticides have achieved good results.

Microbial insecticides may include bacteria, fungi, protozoa or viruses. They may operate through mechanisms such as toxin production [e.g. Bacillus thuringiensis (Bt)] and invasion parasitism (e.g. baculoviruses) (Matten et al. 1993). Although microbial insecticides account for less than 1% of the market, this form of pest control has become the focus of attention in recent years. This is because of the improved performance and cost competitiveness of microbials, the increasing resistance of arthropods to chemical insecticides, and the lack of development of new insecticides (Starnes et al. 1993).

In Taiwan, the use of entomopathogenic fungi to control insect pests has been seen to have great potential since the turn of the last century (Yen 1997). Among different microbial control agents developed and tested, B. thuringiensis, baculoviruses and entomo-pathogenic fungi are considered to be the most promising candidates to control insect pests. Photorhabdus luminenscens with multifunctional activities against plant pests can also be developed as bio-pesticide.

So far, about 1,000 insect pheromones have been isolated, identified and synthesized. Of these, only a handful are used in Integrated Pest Management (IPM) programs in Taiwan including sex pheromones of sweet potato weevil, carambola fruit borer, tobacco cutworm, and beet armyworm.

With the use of serial plot-sealed liquid fermentors, technologies for the mass production of long and stable biomass formulation of microbial fungicides were established. The field trials exhibited satisfactory results against soil-borne diseases.

This paper reviews the recent progress on the development, application and future prospects of bio-pesticides in Taiwan.

Development and Application of Bio-Insecticides

Isolation and Characterization of Entomopathogenic Fungi

A total of 831 fungus-infested cadavers of insects, spiders, and mites were collected from various habitats in different latitudinal zones. From these specimens, 24 genera consisting of 66 species were isolated and identified. Among them, four new species were found, plus one new combination and 37 newly recorded species from Taiwan (Tzean et al. 1997). A parasitic fungus, Neozygites cf. adjarica, was obtained from the spotted spider mite (Shih and Shune 1994). Another investigation was carried out by the Biopesticides Division, Taiwan Agricultural Chemicals and Toxic Substances Research Institute (TACTRI). Results showed that Metarhizium, Beauveria, Nomuraea, Paecilomyces, and Aschersonia were the most common species of entomopathogenic fungi in Taiwan. The survey also showed the occasional occurrence of other entomopathogenic fungi, Verticillium, Gibellula, Hirsutella, and Cordyceps. Results of the API ZYM test showed that all isolates have broadly similar reactions, but the test could be used to distinguish different species. There are differences in pathogenicity among isolates of M. anisopliae, N. rileyi and B. bassiana to the larvae of Spodoptera exigua and Spodoptera litura (Kao et al. 1998).

Random amplified polymorphic DNA (RAPD) was used to differentiate a total of 38 strains of entomopathogenic fungi which were isolated from 20 geographic regions of Taiwan and mainland China. Fungal isolates were obtained from 15 insect species. Banding patterns were generated from three selected primers (OPM 12, 18, and 20). Isolates were grouped into 10 clusters according to similarity, following cluster analysis using Jeffrey's coefficients. Three distinct genotypes were observed among the 38 isolates tested. Based on the RAPD patterns, three species were recognized, namely Beauveria, Metarhizium anisopliae var. anisopliae, and Nomuraea. Nomuraea exhibited a more conservative banding pattern than the other species. RAPD markers may be useful as identification bio-markers of specific bio-control strains in a limited geographical area (Kao et al. 2002).

The optimal temperature for mycelial growth of various Verticillium lecanii isolates was 24°C, but 20°C for isolate F168. V. lecanii isolates were sensitive to higher temperatures. Most isolates could not grow at 32°C, but they grew well in between 16-28°C. On water agar, V. lecanii isolate F168 germinated significantly slower than other isolates with a germination rate of 98% after 16 hours of incubation at 24°C. There was a hundred-fold difference in the amount of spore production among tested isolates when they were incubated at 24°C for nine days. API ZYM test revealed that most isolates had alkaline phosphatase, arylamidase, esterase/lipase, leucine N-acetyl-b-glucosaminidase, acid phosphatase, b-glucosidase, a-galactosidase, naphthol-AS-BI-phosphohydrolase activity. Each isolate showed different chitinase activity on the colloidal chitin-amended medium. Molecular techniques such as AFLP and RAPD of genomic DNA, RFLP of IGS and 18S rRNA, and polymerase chain reaction (PCR) of ITS were used to analyze the relationships among different V. lecanii isolates. The dendrogram produced from the RAPD data in this study divided the 10 tested isolates into two groups. Four local isolates were grouped in the same cluster. The AFLP analysis method provided a lot of information on polymorphism among isolates. AFLP and RAPD techniques could not differentiate Acremonium charticola from V. lecanii isolates. Further, V. lecanii isolates could not be distinguished by IGS-RFLP and ITS sequence (Tsai 2004).

Nomuraea rileyi. N. rileyi was more virulent to Heliocoverpa armigera at 20°C than at 30°C, and had a low LT50 value at all temperatures tested. The optimal temperatures for fungal development in infected larvae were 20 and 25°C. High humidity and more than 12-hour exposure to light are necessary for conidial production on cadavers. Of the eight solid-stage culture materials tested, pig liver as a culture substrate had the highest spore production, i.e. 1.9 x 10 9 conidia/g (Tang 1996). N. rileyi caused 90.5- 100% mortality in the 4 th instar larvae of H. armigera, when 10 7conidia/mL was applied to corn silks and leaves of soybean, tomato and chrysanthemum. Of the 22 pesticides tested, only two fungicides, maneb and propineb, highly inhibited N. rileyi. Field applications of N. rileyi conidial suspension to neonate larvae were found to be as effective as 40.46% carbofuran (EC) at 800-fold dilution in controlling H. armigera (Tang 1996, Tang and Hou 1998). The pathogenicity effect of N. rileyi to S. exigua was studied. The LC 50 values for the 1st up to the 5th instars were determined to be 1.34 x10 6, 1.47 x 10 5, 4.97 x 10 5, 3.56 x 10 4 and 3.1 x 10 5 conidia/mL, respectively. Pupal mortality was 25% when the 5th instar larvae were inoculated with a conidial suspension at 1 x 10 7conidia/mL. The highest mortality rate of the 4th instar larvae infected with N. rileyi conidia was 70%, when sprayed with inoculum from a distance of 2 m away and exposed to a wind blow velocity of 6.0 kg/hr (Tang 1998).

Bioassays in the laboratory showed that N. rileyi was less virulent to younger instars than older ones of the corn earworm or H. armigera, the LC 50 value to the 1st instars being ca. 625 fold higher than that to the 5th. Body surface (mm 2) was positively correlated to larval stage in days (r=0.982). Furthermore, a positive correlation between the conidial loading quantity and the body surface of the instars was observed (r=0.986). Therefore, it is evident that the higher virulent feature of N. rileyi to older instars is due to their larger body surfaces. This permits more conidia on their body, indicating that body surface is an important factor that affects the virulence of N. rileyi to H. armigera. In addition, the higher virulence at the 5th stadium appears to be related to its longer duration, providing sufficient time for conidia to penetrate into the hemocoel to cause mortality. During the 4th stadium, a lower virulence was observed for those larvae close to molting than for the newly emerging ones indicating that ecdysis is an important factor preventing conidial penetration into the hemocoel (Tang et al. 1999).

The effects of environmental factors on infection of the entomopathogenic were significantly different when the inoculated larvae were incubated from 15 to 30°C. The fungal development in inoculated larvae was best at 20 and 25°C after shifting from 20°C to either lower or higher temperatures. The germination rate was higher at 20 and 25°C than at 30 or 35°C. Conidial germination was better on the wash-off of insect cuticle than on Sabouraud maltose agar with yeast extract. Sporulation on chill-dried cadavers reached maximum at 95 or 100% relative humidity than at lower levels of relative humidity on fungus. N. rileyi, isolated from the corn earworm in Taiwan to its host insect, was studied in the laboratory. The fungus caused higher larval mortality at 20°C than at 30°C when 5 x 10 6 conidia/ml were sprayed on the 4 th instar. However, mortality of the 5 th instar injected with 1 x 10 3 conidia/larva was minimal. The time required for sporulation was two days less at 100% than at 95% relative humidity. Although photoperiod did not affect the 5 th instar mortality caused by N. rileyi, their median lethal time (LT 50) values were shorter upon incubating them under light. Incubation of infected cadavers under 12 or 24 h light resulted in 20-fold more conidial production than under full darkness. Therefore, lighting is necessary to develop this isolate on insect cadavers (Tang and Hou 2001).

Response Surface Methodology (RSM) was used to study the optimal media for N. rileyi in submerged cultural fermentation. In a shake flask culture, glucose was found to be the best carbon source, and corn steep powder was found to be the best nitrogen source. The resulting equation from RSM was Y=l.l89+0.0958 X 1,+0.0267 X 2_0.0063 X 3-0.1278 X 1 2-0.1304 X 2 2-0.149 X 3 2. The maximum point of cell production was obtained when the medium contained 3.2% glucose, 29% V8, and 0.5% corn steep powder, and when the dry weight of the cell was 12.1 g/L. When the optimal medium was applied to 5 L of fermentor at 2 vvm, 250 rpm, and 25°C, the harvest of cells was 16.4 g/L which was even higher than that in the shake flask culture (Lin et al. 2003).

The formulation of N. rileyi mainly works on stabilizing conidia activity during preservation as well as eliminating the spore formulation while exposed to UV. The fresh conidia of N. rileyi was heated at 35°C for one hour, then suspended into the soybean oil with 1% glucose. After storage from three to six months, the germination percentage of the formulated conidia decreased from 63% to 46%. With a six-month old oil-based agent, the mortality rate of the 3rd instar larvae of S. exigua remained 63.3%. Four percent of conidia germination was found in the spore powder without formulation after five months of storage. For UV protection, 0.5% zinc oxide was added to the oil-based agent. 74% of germination with 80% mortality of the 3 rd instar larvae was found when exposed under a light of 30 Lux UV for 30 minutes. The spore powder germination without formulation closed to zero (Chien 2004).

A liquid inoculation system was designed and constructed for the mass production of N. rileyi spores. This system was made of aluminum and stainless steel, combined with transport system, pressure supply system, inoculation system, and control system. The inoculation volume was controlled by adjusting spray time and pressure. The maximal inoculation number reached up to 3,600 bottles per hour (Kao et al. 2006). A solid-state fermenter was also invented for the mass production of N. rileyi spores (Kao et al. 2004), as well as an oil-based recovery system for separation and concentration of N. rileyi, M. anisopliae and B. bassiana spores with high efficiency (Kao et al. 2000, Kao et al. 2002).

Metarhizium anisopliae. Field trials were carried out using three types of fungi, M. anisopliae, B. bassiana and N. rileyi, to control S. exigua on Gypophila paniculata and green onion. The M. anisopliae and B. bassiana treatments showed better control of S. exigua on G. paniculata than insecticide. M. anisopliae and B. bassiana were both applied twice a week, for eight weeks, to a green onion field. Results showed that the insect damage level to green onion decreased from 34% to 11% after the fungal treatment. Yields of green onion increased by about 50% (Kao and Tsai 1989). After three applications of M. anisopliae var. anisopliae, formulated as a homogenous biomass in granules or in a conidial suspension, Brontispa longissima could not be detected (Lin et al. 1989). The larval mortality of P. xylostella was 90%, three days after being inoculated with an M. anisopliae MA-126 suspension in a concentration of 10 7conidia/mL. MA-126 wettable powder and liquid formulated preparations were applied, mixed with low dosages of chemical insecticide to control P. xylostella in 120 vegetable production net-houses located in various parts of Taiwan (Lin 1996).

M. anisopliae var. anisopliae MA-805, isolated from the smaller brown planthopper Laodelphax striatellus, is highly pathogenic to its host. Applying it five times to L. striatellus and once to the brown planthopper Nilaparvata lugens enhanced its virulence (Lee and Hou 1989a,b). Six of 12 pesticides tested (hymexzel 30 L, benomyl 50 WP, tricyclazole 75 WP, pendimethalin 34 EC, paraquate 24 EC, and alachlor 45.1 EC) suppressed the mycelial growth of M. anisopliae var. anisopliae NA-805 (Lee and Hou 1989c). Of the four insecticides tested, chlorpyrifos had the strongest inhibitory effects on M. anisopliae var. anisopliae Ma2. Mixing methomyl at concentrations recommended for field applications with Ma2 spore suspensions had a synergistic effect on S. exigua larvae, reducing its damage to leaves and the period before larvae die (Tsai et al. 1993b).

Benomyl- and carbendazin-resistant isolates (UV-Be, UV-Ca, Mu-Be, Mu-Ca) of M. anisopliae var. anisopliae were obtained when conidia were treated with ultraviolet light or mutagenic agents. These isolates had cross resistance, and showed stable fungicide resistance. A progressive increase in benomyl and carbendazin concentrations on culture media could also induce the appearance of fungicide-resistant isolates (Fe-Be and Fe-Ca) (Tasi et al. 1993a). Fungicides (benomyl, carbendazin, curzate, metalaxyl, and mancozeb) were highly toxic to M. anisopliae var. anisopliae Ma2 isolate. Although benomyl and carbendazin are chemically similar fungicides, the former is fungicidal to Ma2 while the latter is not (Tsai et al. 1994).

Mass production of M. anisopliae var. anisopliae MA-805 was carried out using grain and plant residues as substrates. It was found that coarse rice, corn cob and bagasse were effective substrates for sporulation of this fungus (Lee and Hou 1989c). Kao and Tsai (1989) and Kao et al. (1989) found that conidia could be mass produced with steamed, polished rice at 28°C, with a 24-hour photoperiod. Rice grain, soybean, and other agricultural by-products such as rice bran and rice steep were used as additives to formulate solid fermentation culture media for spore mass production. Yeast extract, dextrose and Sabourand broth were used in submerged fermentation for mycelia production (Liu 1996).

M. anisopliae var. anisopliae (Ma2) was used for destruxin production. Results showed that when 30 g/L bactosacharose was used in submerged fermentation, the destruxin yields were 1.57 mg DB, 1.16 mg DMDB, 0.34 mg DA, and 0.14 mg DE. With solid-state fermentation, it was found that 100 g of rice (dry weight) yielded 4 mg DA, 3.03 mg DMDB, 2.29 mg DB, 0.95 mg DE and 0.21 mg DA2, and produced 2 x 10 9 spores per gram of rice (Hsieh et al. 1998a). An extract of solid-state fermentation from Ma2 showed a high level of virulence to S. exigua. For the 3 rd instar larvae, the LC 50 values of the original extract were 1.41 x 10 5 ppm, 7.07 x 10 4 ppm, and 5.18 x 10 4 ppm, after one, two and three days of infection, respectively. Among the fractions separated by a silica gel, the fractions containing 32% DA, 31% DB, and 4.5% DE had virulence equal to that of the original extract (Hsieh et al. 1998b).

Beauveria bassiana. B. bassiana was isolated from Taiwanese soils infested with C. formicarius (Su 1991a,c, Su et al. 1988). Field trials showed that spraying with 1.6 x 10 4 conidia/mL at the time of planting or rootstock formation, or broadcasting soybean seeds containing 10 9conidia/g at planting time, effectively controlled the weevils (Su 1991a). B. bassiana was pathogenic to 3rd, 4th and 5th stadium nymphs and adults of Riptortus linearis. At 25°C and above, pathogenicity of B. bassiana decreased as temperature increased. UV irradiation of conidia reduced the pathogenicity of B. bassiana to R. linearis (Hu et al. 1996). Under open-air conditions, three formulations could be produced: 1) a solid-state soybean substrate inoculated with blastospores; 2) a granulated form of the B. basiana-soybean mixture (BSM); and 3) a powdered form of B. bassiana-soybean powder (BSP). Their spore concentrations were ca.1 x 10 8 -10 9 and 1 x - 10 9 -10 10 conidia/g, respectively. The pathogenicity of BSP was different to that of B. bassiana rice powder. The LC 50 values to O. furnacalis of the two formulations were 4.8 x 10 6 and 9.5 x 10 7, respectively (Chiu 1989).

Corn plants at the late whorl stage were infested with ten 2 nd instar larvae of O. furnacalis, and then treated with 2-3 g of BSM two days after larval release. Only 0.19 larvae/plant and 13% infestation were found in treated blocks which were surveyed two weeks after treatment. Four weeks after treatment, there were 0.36 larvae/plant and 27% infestation. This can be compared to the 4.58 and 4.26 larvae/plant and 100% infestation recorded in untreated blocks (Chiu 1989). B. bassiana was able to infect the eggs, larvae and pupae of O. furnacalis. When cultured on wine derivatives and mixed with sand to form granules at 2 x 10 8 conidia/g, B. bassiana was as effective as carbofuran in screenhouse tests (Chiu and Hou 1993).

Liaphis erysimi Kalt treated with B. bassiana at a concentration of 107 conidia/mL showed almost 100% mortality. By the third day, B. bassiana was able to grow at temperatures ranging from 10°C to 30°C in a yeast-peptone-dextrose medium (Hsiao and Lin 1995). The effect of pesticides on this fungus was studied in the laboratory. Results showed that mycelial growth was completely inhibited by the fungicide Sporatak (Su 1988). Zineb, iprodione, metalaxyl+mancozeb (MMC), metalaxyl+copper oxychloride (MCO), thiophenate methyl+streptomycin (TMS), propineb and imazalil were detrimental to B. bassiana at certain concentrations tested. Bupirimate had the smallest inhibitory effect on the fungus. Imazalil showed complete inhibition in liquid culture, irrespective of the length of time the fungus was treated (Hsiao and Lin 1995).

The DNA sequence of PCR-amplified gene fragments from different Beauveria spp. isolates, using a PI-P3 primer set, was examined. Results showed more than 95% homology among the isolates from Taiwan and one isolate from mainland China. However, the PCR amplification profiles of Beauveria spp. isolates using 18S rRNA primer set varied greatly. This can be used to characterize and identify isolates of Beauveria spp. (Shih et al. 1998).

Two local isolates of B. bassiana (Bb Col. 41 and 42) and one isolate of V. lecanii (HO 159) were bioassayed against turnip aphid (L. erysimi). Seventy-five newly molted apterous from stock colony were randomly selected for each bioassay and exposed to the fungal pathogens placed for 15 minutes. Treated and non-treated aphids were then transferred individually into a 5.5 cm (dia.) petri disc and assigned each aphid as a replicate. Detached leaves from four-week old and non-heading Chinese cabbage (Brassicae chinensis) plants were used as the food source. Treated and untreated aphids were placed at 20°C and 25°C incubators with a photoperiod 12 L: 12 D. The mortality and fecundity of turnip aphids were recorded daily. The outgrowth and sporulation of these three fungal isolates on aphid cadavers after treatment were also recorded using a ranking system. The average fecundity in infected and controlled aphids differed significantly at 20°C and 25°C after three days inoculation. The average fecundity of infected aphids for Bb-infected apterous female were 4.0 and 4.2, and 4.4 for Vl-infected, while for the control, 17.2 per aphid at 25°C was used, wherein the corresponding fecundities were 1.2, 1.6, 2.3 and 14.3 at 20°C, respectively. Significantly more sporulation was found on VI when compared with other two fungal isolates at 20°C, while there was a significantly greater sporulation on Bb Col.41 at 25°C. The liquefaction of cadavers infected by Bb Col. 42 was observed which might be responsible for the less sporulation of turnip aphid cadavers (Hsiao and Lin 1995).

Verticillium lecanii. V. lecanii could not grow at a temperature as high as 35°C. For V. lecanii F173, maximum growth and sporulation was seen at 25°C. The highest sporulation was shown when F173 was grown on yeast-peptone-dextrose medium. For both F159 and F113, the optimal radial growth and maximum sporulation were observed at 25°C. The highest sporulation was evident when fungus was cultured on Sabouraud's dextrose agar, but in general, isolates of V. lecanii grown on yeast-peptone-dextrose medium showed better growth and sporulation (Hsaio and Yang 1998). Of the eight fungicides tested, propineb, imazalil, and matalaxyl+mancozeb were fungicidal to V. lecanii. Buririmate, zineb, thiophenate methyl+dytrpyomyvin and iprodione were moderately inhibitory to mycelial growth and sporulation of V. lecanii. On the other hand, of the insecticides tested, insecticidal soap and abamectin had relatively little adverse effect on the mycelial growth and sporulation of V. lecanii (Lin et al. 1998).

V. ecanii was isolated from Rhopalosiphium padi and was reported to be highly pathogenic to Myzus persicae, Macrosiphoniella sanborni, Toxoptera aurantii, L. erysimi, Aphis gossypii, and Saissetia oleae. The cultural characteristics and pathogenicity of the fungus were very stable after 13 subcultures on Sabouraud's medium (Peng 1985). Other bioassay tests also showed that V. lecanii isolates at a concentration of 1x 107conidia/mL were highly virulent to L. erysimi, M. persicae, and Thrips palmi. It caused 60% mortality of T. palmi after five days of treatment, while the mortality of L. erysimi was 90%. In contrast, V. lecanii was not pathogenic to A. gossypii. Conidia of V. lecanii isolates germinated when relative humidity was more than 94%. Conidial germination was affected by momentary drought and high temperature. Conidia of V. lecanii isolates germinated and penetrated directly into M. persicae without producing appresorium. Most conidia could be found around setae (Tsai 2004).

A strong harmful effect of UV irradiations on conidial germination of V. lecanii isolates was observed. Exposure to UV-B irradiation (310 nm, 540 mW cm -2) or UV-C (254 nm, 120 mW cm -2) for two minutes postponed the germination of three isolates tested (F096, VL159 and VL578). This delayed effect did not occur when the conidia were exposed under UV-A irradiation (360 nm, 540 mW cm -2) for 90 minutes. A UV-induced dimer, cyclobutane pyrimidine dimer, could be detected by using T4 endonuclease digestion method. Among 10 UV protectants tested, uric acid, folic acid, active carbon and xanthine provided significant UV-protective effect for V. lecanii. Active carbon was the best UV protectant among them. Molasses or plumbago also possessed the UV-protective effect for the conidia of V. lecanii. The feasibility of V. lecanii isolates to control aphid on cabbage was investigated. A field trial showed that weekly spray of indigenous V. lecanii isolates could control aphid when the population density was below 20 aphids per leaf. (Tsai 2004).

Isolation and Characterization of Baculoviruses

Virulent baculoviruses have been isolated from lepidopterous insects. Tests carried out in both the laboratory and the field gave encouraging results. The study used five nucleopolyhedro-virus (NPV) isolates from Taiwan, namely, Spodoptera exigua nucleopolyhedrovirus (SpeiNPV), Spodoptera exigua NPV (SpltNPV), Perina nuda NPV (Penu NPV), Lymantria xylina NPV (Lyxy NPV), and an isolate of Autographa californica NPV (AcMNPV-TWN4) from S. exigua. The EcoR I profiles of genomic DNAs of five NPVs gave a clear pattern for identification. A set of primers designated as primers 35 and 36 (Chou et al. 1996) was used for amplification of polyhedrin gene from five NPVs by PCR, with 680 bps of amplicons. These polyhedrin gene fragments were sequenced and were then digested with BsuR I, BspH I, BsiW I, Hpa II, Mse I and Taq I DNA restriction endonuclease, respectively. It is suggested that PCR-RFLP technique is a feasible method for rapid detection and identification of five NPVs isolates from Taiwan (Kao et al. 2000).

Artogeia rapae granulosis virus (ArGV) and Plutella xylostella granulosis virus (PxGV). The application of ArGV, PxGV virus, and S. litura nuclear polyhedrosis virus (SpltNPV) gave effective control of A. rapae, P. xylostella and S. litura, respectively. The use of mixtures of ArGV and PxGV, ArGV and SpltNPV, and PxGV and SpltNPV resulted in the effective control of A. rapae and P. xylostella, A. rapae and S. litura, and P. xylostella and S. litura, respectively. The combination of two GVs and one NPV was also effective in controlling P. xylostella, A. rapae and S. litura (Su 1989).

Spodoptera litura nuclear polyhedrosis virus (SpltNPV). The 3 rd instar larvae treated with 10 x 10 6 PIBs/mL stored for four years showed a mortality of 83.3%. Larvae given the same treatment at the same concentration, but stored for only half a year, showed 96.7% mortality, while a fresh virus showed 100.0% mortality. S. litura applied with a combination of SpltNPV and palcrasol As-29 had 87.1% mortality. A strong spread gave 83.2% mortality, and CS-7 70.6% or SpltNPV alone possessed 75.8% mortality (Su 1992).

This SpltNPV isolate was highly pathogenic to S. litura larvae. The LC50 values were 5.47 x 10 5, 4.47 x 10 4, 6.16 x 10 5, 3.12 x 10 6, 1.4 x 10 7, and 7.28 x 10 8 PIBs./mL from the 1st to 6th instar, respectively, as assayed through the inoculum-imbibing method. Mortality rates were higher in older larvae, but were also slower to take effect (Tuan et al. 1995a). The virus was more thermo-stable as a dry powder than as a suspension. The virus retained more than 80% of its original activity when exposed at 55°C for 24 h. SpltNPV in a suspension at 5, 7, and 9 pH for 24 h was not significantly affected. However, exposure to a pH of 10 and 11, or to solutions containing chloride ions at high concentrations, greatly weakened its effect. Uric acid provided SpltNPV significant protection from sunlight (Tuan et al. 1995b). It is suggested that SpltNPV is well suited for mass production. An ideal method is to use it on as early as the 5 th instar larvae, by individually incorporating the isolate into their diet (an inoculum of 3 x 10 6 PIBs/mL diet) and incubating them for seven days at 30°C. The average yield was 1.4 x 10 9 PIBs/larva. Standardization and quality control of SpltNPV products can be achieved by visual counting, bioassay, SDS-PAGE, and ELISA. Application of SpltNPV at a low concentration on egg masses immediately before hatching resulted in 77.4% larval mortality, compared with 50.4% on newly laid egg masses. Applications of SpltNPV at high concentrations (10 8 PIBs/mL) resulted in 99.2% larval mortality, and a 94.25% decrease in the area of leaves eaten. The control efficacy of SpltNPV at high concentrations was better than that of bifenthrin with Bt one week after application (Tuan et al. 1998).

The activity of SpltNPV against S. litura larvae was enhanced by mixing the virus suspension with leucophor, fluorescent brightener 28, lecithin, phosphatidyl choline, and phosphatidyl ethanolamine. The mortality of the S. litura larvae increased with higher concentrations of the chemicals in the virus suspension, with the exception of the fluorescent brightener 28. Of the six Granulosis viruses tested, the PxGV enhanced the effectiveness of SpltNPV the most (Lee, 1996).

Heliothis nuclear polyhedrosis virus (HsNPV). H. armigera densities were significantly reduced 14 days after application of HsNPV. Control was better than that achieved with carbofuran. Injecting HsNPV preparations into corn ears was more effective than spraying it (Tuan et al. 1989a). The HsNPV was inactivated by the weakly alkaline dew (pH 8.1) collected from soybean leaves. Heavy artificial rainfall of 242 mm/h for 30 minutes failed to wash off HsNPV preparations sprayed onto corn silks (Tuan et al. 1989b). Lecithin added to an NPV commercial product reduced LT50 values by two to three days, compared to inoculation with NPV alone (Tuan and Hou, 1998).

Spodoptera exigua nuclear polyhedrosis virus (SpeiNPV). A SpeiNPV isolate was obtained from S. exigua larvae that originated from G. paniculata in Taiwan (Kao et al. 1991a,b). This SpeiNPV isolate is highly pathogenic to S. exigua larvae. Its LC 50 values were 1.6 x 10 6, 6.9 x 10 4, 4.1 x 10 4, 3.7 x 10 5, and 5.2 x 10 6, PIBs/mL for the 1 st to 5 th instar, respectively, as assayed using the inoculum-imbibing method (Tuan et al. 1994). Data on food consumption, virus yield, larval mortality and microbial contamination suggest that SpeiNPV is well suited to mass production using an artificial diet of pellets each weighing 1.54 ± 0.23 g (diameter 1.0 cm, height 0.5 cm), containing an inoculum rate of 2 x 10 5 2 at 30°C, and are harvested 5-6 days after inoculation (Huang and Kao 1994).

Bioassay demonstrated that of the materials tested, 15 adjuvants increased the effectiveness of SpeiNPV. Bivert, agral 90 and nu-film 17 were the most effective (Kao et al. 1991a). Results also showed that the addition of 1% uric acid, 1% activated carbon, 1% folic acid or 1% xanthine to the viral suspension provided significant UV protection for SpeiNPV (Kao and Huang 1992).

Field trials showed that good control of S. exigua in green onion was achieved at a rate of 1.5-2.0 x 10 12/ha, with Bivert as a spreader-sticker. Twenty hectares of green onion were sprayed with SpeiNPV to control S. exigua in 1996, with 67 farmers participating. The demonstration of this control strategy received favorable comments from the participants. In 1997 the project was expanded to 200 ha with 213 farmers participating (Kao et al. 1997).

Autographa California nucleopolyhe-grovirus (AcMNPV). Trichoplusia ni, P. xylostella, and S. exigua were highly susceptible to AcMNPV. LC 50 values of AcMNPV to 3rd instar larvae on the sixth day after inoculation were 1.24, 2.07 x 10 3, and 2.40 x 10 3 PIBs/mm 3. Heliothis virescens, S. litura, O. fumacalis and Galleria mellonella were found to be moderately susceptible to AcMNPV. H. armigera showed a very low mortality rate, while Corcyra cephalonica was no infected with AcMNPV (Tuan et al. 1997).

The d-endotoxin gene from Bt aizawai 7.29 was inserted into AcMNPV. The recombinant viruses Acendo-T7, Acendo-Uwl and Acendo-1993 were purified by end point dilution, and identified through dotblot hybridization, the PCR method and southern blotting. Results showed that the expression of the ä-endotoxin gene using the AcMNPV expression system improved the cytotoxicity of this virus (Hu et al. 1994).

For improving the UV resistance of AcMNPV, the melanin gene from Streptomyces lividans was cloned to the AcMNPV transfer vector pVL1393. The recombinant transfer vector DNA was cotransfected with parental viral DNA into a S17B cell line derived from S. litura. It showed that the recombinant virus, Actyr, contains a melanin gene. The expression of protein was checked by SDS-PAGE. The data level of protein expression increased over time after viral infection (Lin 1995).

Jinn et al. (Jinn et al. 2005) employed the internal ribosome entry sites (IRES) element of encephalomyocarditis virus (EMCV) to generate a red and green dual fluorescence protein gene containing recombinant AcMNPV named vAcR-IR-G, to evaluate the efficacy of fluorescence proteins as recombinant AcMNPV tracer. vAcR-IR-G that infected SF9 cells emitted red fluorescence as well as green fluorescence and indicated that the EMCV-IRES can mediate cap-independent translation in SF9 cells. However, the EMCV-IRES did not function well in insect larvae infected with this recombinant AcMNPV. Interestingly, it is found that red fluorescence protein from Discosoma sp., DsRed, is as an excellent marker as the recombinant AcMNPV tracer. Insect larvae, including Trichoplusia ni, Spodoptera exigua, and Spodoptera litura, infected with the recombinant AcMNPV containing the DsRed gene can emit red fluorescence under visible light. Thus, the red fluorescence protein from coral will facilitate the genetically modified development of baculovirus

An attempt to demonstrate the insecticide activity of boric acid on AcMNPV and SpeiMNPV was carried out in the laboratory. Results showed that boric acid significantly enhanced the insecticide activity of AcMNPV and SpeiMNPV and acted as an effective viral synergist (Jinn et al. 2004).

Bacillus thuringiensis (Bt). B. thuringiensis (Bt) has been registered for use against several lepidopterous pests of vegetables in Taiwan since 1960. P. xylostella (diamondback moth), the most destructive pest of cruciferous crops, has developed resistance to a number of synthetic insecticides in Taiwan (Sun 1992). As a result, farmers became interested in the use of Bt to control the moth. Research on novel Bt strains, genetic engineering, enhancement of efficacy, optimization of the growth medium, field trials and quality control have been carried out by a number of research institutes and universities in Taiwan.

Isolation and Characterization of Local BT Isolates

Hundreds of Bt isolates have been isolated from different parts of Taiwan. In addition to plasmid profile and western blotting, PCR was also used to characterize Bt isolates (Chak and Jen 1993, Chak and Young 1990, Chak et al. 1994, Chak et al. 1995, Chao 1992, Chen 1992, Kao et al. 1994, Li 1991). Using a set of specific oligoprimers for PCR amplification, Chen (1992) found that the Bt YMB 96 strain contained cry1Aa and cry1Ac genes. Chao (1992) detected six kinds of cry1 gene types residing in his Bt isolates.

Four different techniques were employed to isolate Bt from stored materials collected from around Taiwan. Four hundred and eighty isolates were obtained. PCR analysis was applied to identify isolates harboring different cry-type genes. Thirty-eight distinct cry-type gene profiles were detected. The cry-genes included cry1Aa, 1Ab, 1Ac, 1B, 1C, 1D, 1E, 1F and cry1. Agarose gel electrophoresis and SDS-PAGE were used to characterize plasmid and protein profiles for each isolate. Results showed significant differences in size among the isolates. Diet incorporation technique was undertaken to evaluate the bioactives of each isolate. Bt isolates Al-9, G2-1 and G3-3, containing cry1Ab, 1B, and cry1, exhibited the most insecticidal activity against P. xylostella (Kao et al. 1996).

Two pairs of universal oligonucleotide primers designed by Kuo and Chak (1996) were used to probe the best conserved regions of all known cry1-type gene sequences, so that the amplified PCR fragments of the DNA template from Bt isolates would contain all possible cry1l-type gene sequences. The RFLP patterns of PCR-amplified fragments revealed that 40 distinct cry-type genes have been identified from 146 Bt isolates from stored-grain warehouses in Taiwan. Of these, 29 cry-type genes were found to be possibly new, including cry1lAc *1-5, cry1C *1, cry1Cb *1-4, cry1D *1-4, cry1E *1-7, cry1F *1-2, cry1G *1, cry1Cb *1 or cry1D *1 or cry1F *1, and unknown *1-4. However, the novelty of these cry-type genes needs to be confirmed (Kao et al. 2003).

A new cry gene (cry1Ca9) was cloned and sequenced from an isolate (G10-01A), harboring possible novel cry1C gene. The cry1Ca9 gene consisted of an open reading frame of 3,567 bp encoding a protein of 1,189 amino acid residues. The polypeptide has the deduced amino acid sequences predicting molecular masses of 134.7 kDa. When the cry1Ca9 toxin was expressed in cryB-, a non-enterotoxigenic and non-cytotoxic plasmid negative Bt strain, elliptical crystals were produced. The recombinant strain exhibited high toxicity against Plutella xylostella (Kao et al. 2003). A cry1Ac gene was cloned and sequenced from an isolate E9-11. This gene was consisted of an open reading frame of 3.6 kb. The polypeptide has deduced amino acid sequences predicting molecular masses of 130 kDa. When the cry1Ac toxin was expressed in cryB-, spherical crystals were formed. The recombinant strain showed similar toxicity to that of Xentari, Delfin, and HD-73 against P. xylostella. However, it is more toxic against larvae of Arotogeia rapae than all other samples tested (Tzeng et al. 2002).

Genetic engineering of Bt. The epiphytic Erwinia herbicola has been transformed with cry1Aa1 presented in plasmid pUN4. Protein extracts from this transformant exhibited strong toxicity against P. xylostella. The transformed E. herbicola also showed significantly antagonistic effects against Xanthomonas campestris pv. campestris and E. carotovora subsp. carotovora. This result firstly demonstrated the combinations of insecticidal activity, and the characteristics of antagonism and foliar colonization in the same transgenic epiphytic bacteria (Lin et al. 2003).

Enhancement of Bt efficacy. The addition of 0.1% octyl methoxy cinnamate to Bt preparations was found to prolong the persistence of their toxicity to P. xylostella by 1.62 times. Starch, sugar and 0.05% methyl paraben mixed with the Bt preparations slowed down the rainfall wash-off from rape leaves, and increased Bt efficacy by 4.2 times. Incorporation of over 0.1 ppm sinigrin into low-dosage Bt preparations doubled the insecticidal efficiency, which then increased eight times on the 4th instar larvae if sinigrin was added at 1 ppm (Chung 1991).

Bt formulations were prepared by: a) blending predetermined amounts of pregelatined starch, natural starch, Bt and water to form a mass; b) drying this mass by heating with a hot roller at temperatures between 50°C and 100°C, (preferably between 60°C and 90°C), to form a dried mass; and (c) crushing the dried mass to form the final product in the form of flakes, which should have a diameter of less than 900m. Using this method, the processing time was reduced from 24 hours to less than 30 minutes, or even 3-15 minutes (Yang et al. 1994).

A biocidal formulation with improved resistance to ultraviolet rays is made up of: a) Bt; b) an anti-ultraviolet agent selected from the group consisting of 2-(2-H- benzotriazole-2-yl)-phenol and their derivatives; c) pre-gelatinized starch; and d) natural starch. The anti-ultraviolet agent, the pregelatinized starch and the natural starch are mixed then baked at 60-90°C to form the Bt formulation (Yang et al. 1995).

Optimization of growth medium. RSM was used to assess the impact of the composition of various cultivation media (tapioca, fishmeal, CaCO 3 and (NH 4) 2SO 4) on the growth of Bt YMB 96-1988. Estimated optimum compositions for the production of spores by Bt were as follows: tapioca, 5.01%; fishmeal 5.86%; (NH4) 2SO 4 0.06%. This mixture gave a maximum spore count of 8.56 x 10 8/mL. This value is close to the 8.35 x 10 8/mL spore density as counted from experimental observations (Liu and Tzeng 1998).

Characterization of the sporulation of B. thuringiensis. A wild-type and an rDNA sstrain of B. thuringiensis were cultured in a net-draft-tube modified 20-L airlift bioreactor. A comparison of the sporulation patterns suggests that the early sporulation strain has a lower final spore count. Results from off-gas analysis suggest that the CO 2 profile could be an alternative indication to spore counts to examine fermentation performance or even the mortality in bioassay of the cultivation product. The difference in mortality tests exhibited by the microorganism was attributed to different patterns of sporulation as well as different levels of gene control inside the cell itself. The sporulation kinetics of Bt was simulated through a simple modified Hill equation, where the initial glucose concentration could affect the timing of the onset of sporulation. The equation matches well with the experimental sporulation data for Bt in both wild-type and rDNA strains (Lin and Tzeng 2000).

Field control of P. xylostella, Artogeia rapae and Ostrinia furnacalis with Bt. Results obtained from field trials for pest control were satisfactory. The use of Bt was effective in controlling both P. xylostella and A. rapae within seven days of application (Su 1991c). Results of two field experiments indicated that new commercial Bt products gave better control against P. xylostella than older ones. All Bt products showed fair control of A. rapae (Kao et al. 1990). Chiuo) 1990) confirmed that combining Bt kurstaki with B. bassiana enhanced its effectiveness against O. furnacalis.

Quality control of Bt products. A standardized bioassay protocol has been developed to evaluate preparations derived from Bt against P. xylostella. It has now been adopted by the government as a guideline for Bt product registration (Kao and Tuan 1992). The ELISA detecting systems established by polyclonal antibodies was developed for Bt d-endotoxin (Tuan et al. 1993). SDS-PAGE was also developed to quantify d-endotoxins (Tuan et al. 1993). Application of the ELISA and SDS-PAGE to monitor the d-endotoxins with bioassay was also examined. Those assays were found to be useful, not only in fundamental research and product analysis, but also in the regulatory control of Bt (Kao and Tuan 1995, Tuan et al. 1993). An in vitro assay, devised with cultured P. xylostella cells in agar plate and trypan blue layered on the upper surface, rapidly detected the toxicity of Bt ?-endotoxins (Lee 1993).

Photorhabdus luminescens. Supernatant fluid of Photorhabdus luminescens culture was centrifuged, filtered, and bioassayed against six species of insect larvae. The LC 50 values of protein preparations against the 3 rd instar larvae of the lepidopteran Plutella xylostella amd Galleria mellonella were 56 and 200 ppm, respectively. Antagonistic effects of bacterial culture and protein preparations on 10 species of fungi and nine species of bacteria were screened through dual or concomitant culture methods. High antimicrobial activities were observed against Botrytis cinerea, Glomerella cingulata, Alternaria mali, Phyophthora capsici, B. thuringiensis, B. subtilis, B. cereus, and Erwinia carotovora subsp. carotovora. In conclusion, P. luminescens ATCC 29999 has high insecticidal and antimicrobial activities against local pests and pathogens (Hsieh et al. 2004).

An entomopathogenic nematode, Heterorhabditis brevicaudis TG01, was isolated from sampled soils for the first time in Taiwan using the Galleria-bait method. Identification of the nematode was mainly based on observations under scanning electron microscopy (SEM) and nucleotide sequence of the internal transcribed spacer 1 (ITS1). P. luminescens subsp. akhurstii was isolated from nematodes and identified by phenotypic, biochemical tests, 16S rRNA and Biolog identification system. In this study, the supernatant fluid of the bacterial culture was centrifuged, filtered, and bioassayed against five key pests in vegetables. It exhibited insecticidal activity against Plutella xylostella. Antagonistic effects of bacterial culture and protein preparations on 18 species of fungi and 12 species of bacteria were examined by dual or concomitant culture methods. High antimicrobial activities against Glomerella cingulata, Colletotrichum musae, Xanthomonas spp. and Erwinia spp. were observed. Studies were also carried out to test if mango anthracnose can be prevented during storage with local P. luminescens and consequently showed satisfactory results. (Hsieh et al. 2007).

Pheromones and Attractants

The beet armyworm, tobacco cutworm, sweet potato weevil, citrus leaf miner moth, litchi fruit borer, green peach aphid, sugarcane wireworm, carambola fruit borer, oriental fruit fly and medlon fly are serious insect pests in Taiwan. New and improved synthesis methods and results of the isolation and identification of the above-mentioned 10 insect pests have subsequently been unveiled. Yields of synthesized pheromones can he efficiently increased, and the costs for producing those pheromones can also be drastically reduced by the new, simple and convenient methods developed.

The synthesized sex pheromones for the beet armyworm, tobacco cutworm, sweet potato weevil and sugarcane wireworm have proven to be powerful attractants for each respective species, and were widely applied in the fields to control them. While synthesis of the sex pheromone of the litchi fruit borer was complicated, it can be used for species identification, as well as for monitoring the proper schedule of its application. Large-scale application of the sex pheromone of the carambola fruit borer was used in different ways of monitoring, mass trapping, and mating disruption. The unique property of the aphid alarm pheromone can be used to enhance the effectiveness of insecticides against aphids. (Yen et al. 2004). Examples of the utilization of sex pheromones are given below.

Sex pheromone of sweet potato weevil, Cylas formicarius elegantulus (Summers). A modified and reliable method of synthesizing (Z)-3-dodecen-1-01 (E)-2-butenoate, the sex pheromone of C. formicarius elegantulus (Summers), has been reported. The effective bioactivityof the pheromone synthesized by this new method has also been demonstrated (Yen and Hwang 1990). An alternative method of synthesizing this sex pheromone without using carcinogenic etyhene oxide and HMPA has also been described. C. formicarius was strongly attracted to the synthetic sex pheromone (Lo et al. 1992). Results showed that the funnel type polyethylene terephalate (PET) bottle trap, baited with 1mg of synthetic sex pheromone dispensed in polyethylene, was the most effective, cheapest and easiest way to mass trap male weevils (Hwang et al. 1989, Hwang et al. 1991).

Sex pheromone of the carambola fruit borer, Eucosma notanthes Meyrick. Results showed that the use of this pheromone in double plexiglass boxes appeared to be a successful method of bioassay (Hwang et al. 1996). 0.5-1 mg Z-8-dodeceny1 acetate dispensed in a rubber septum was the most effective and long-lasting formulation for capturing male moths. It was also easy to prepare. A funnel-type triple PET bottle trap with 16 openings of 0.6-0.8 cm each, and with the lure placed 5 cm above the openings, was an effective trap for male moths (Hwang and Hung 1994). According to the results, when disruptant was used in a rubber septum, its strength lasted for five months. The number of damaged fruits was reduced to 10.8-71.6%. These findings revealed that the application of synthetic sex pheromone in the field could be an effective and safe technology for the control of E. notanthes (Hwang and Hung, 1997a.b.).

Sex pheromone of tobacco cutworm, Spodoptera litura (F). A new route to synthesize the major component of the sex pheromone of (Z,E)-9, 11-tetradecadien-1-yl acetate [(Z, E)-9, 11-TDDA] was proposed. The final production (Z, E)-9, 11-TDDA) was 12.8g, with the isomerically pure product of 86.0% (Lo et al. 1988). Partially purified synthetic [(Z, E)-9, 11-TDDA, 86% active isomer)] mixed with purchased 9.12-isomer (95% active) in a ratio of 10:1 could attract male moths of S. litura in field tests. The PET bottle trap was good for trapping male moths (Lo et al. 1989, Lo et al. 1998).

Sex pheromone of beet armyworm Spodoptera exigua Hubner. A method has been developed in synthesizing two major components of the sex pheromone of S. exigua Hubner, namely, (E,E)-9, 12-tetradecadien-1-yl acetate [(Z, E)-9, 12-TDDA] and (Z)-9-tetradecenol [(Z)-9-TDOL]. Results showed that combination of the synthesized components was effective, and its effectiveness could last up to two months in the field (Yen et al. 1998).

Development of Microbial Fungicides (Tzeng and Yeh 2003; Ftzeng Et Al. 2004)

For the establishment of mass production techniques, a plant equipped with 5L to 750L series liquid fermentors and other necessary facilities was constructed. Using Streptomyces saraceticus SS31 isolate and Bacillus subtilis BS1 isolate, each as the model isolate, liquid fermentation protocols for the mass production of biomass formulation were established.

For S. saraceticus SS31, the established protocol yielded approximately a 4 x 10 10 cfu/ml culture broth that contained mainly mature bacterial spores. The liquid formulation produced could remain viable for more than 10 months when stored at 6°C and was effective in controlling various fungal infections that included diseases on various crop species caused by Pythium spp., Rhizoctonia solani, Phytophthora parasitica, Fusarium oxysporum, and Colletotrichum gloeosprioides.

Whereas for B. subtilis, liquid fermentation yielded approximately 5 x 10 9 cfu/ml and the bacterial propagules remained viable for more than one year when stored at 6°C. The application trials conducted indicated that the cultural broth was effective in stimulating plant growth, aside from discouraging infection of various fungal and bacterial pathogens. Notable protective effect covered diseases caused by Xanthomonas spp., Sclerotium rolfsii, and Cereospora nicotiana.

The two sets of liquid fermentation protocols were found to be easily adaptable for the production of members of antagonistic B. subtilis and Streptomyces spp., each with morphological characteristics that closely resemble that of the respective model isolate. One of the major breakthroughs of the technology development was the manipulation leading to the maximized yield of bacterial spores with long durable shelf life. Regarding the commercialized use of Streptomyces spp. for plant disease control, this is the first success in mass production of long durable spore formulation.

The technology platforms were further improved and yielded over 1011 cfu/ml spore biomass for Streptomyces griseobrunenneus S3, over 10 11 cfu/ml endospore biomass for B. subtilis WG6-14, and over 10 8 cfu/ml of chlamydospore biomass for Gliocladium virens WJGV2. The broth cultures obtained can be directly used for field application or further processed into powder or granule formulations; they can be applied by foliar spraying, soil drenching, and seed soaking/coating, like in chemical pesticide application. The application of broth formulations for all three target microbes has shown growth promoting effect and was effective in controlling the diseases selected. The effectiveness of disease control was comparative to that by chemical fungicides especially for the control of soil borne diseases.

Conclusion and Future Perspective

It is likely that insect management through synthetic organic chemicals will still dominate the world pesticide market. However, major thrusts are under way in industrial, academic, and government research laboratories to develop more selective methods of pest control which are compatible with biological, toxicological, environmental, and societal requirements (Altstein et al. 1993). In Taiwan, environmental concerns and insecticide resistance have recently led many farmers to consider the use of microbial insecticides for the control of agricultural pests. The following topics are particularly worthy of more attention:

Isolation, Screening and Strain Improvement of Indigenous Entomopathogens

Taiwan's subtropic and tropic climate, diversified topography and mosaic cropping systems provide rich microbial resources. There is good potential for finding novel agents with high pathogenicity and broad spectra. The selection of effective strains of entomopathogens, and microbial breeding and genetic manipulation to improve strains, are essential to the development of microbial insecticides (Aizawa 1987).

Development of Microbial Control Strategies in Ipm

Microbials are ideal for use in integrated pest management (IPM) because of their selectivity and environmental safety (Starnes et al. 1993). To maximize the efficiency and ecological compatibility of microbial insecticides, specific methods of application should be designed for their use.

Improved Production and Formulation

The success or failure of any microbial insecticide depends mainly upon the technology of its mass production. It is essential that a stable strain be selected, with high pathogenicity and productivity. This will optimize culture conditions and cut down production costs.

Opportunities for enhancing field effectiveness and prolonging the shelf-life can exist through improved formulation. Suggested areas for advancement include micro- encapsulation biotechnology, and the use of various additives such as stickers, spreaders, and feeding stimulants, as well as UV protectants.

Enhancement of Molecular Biology and Genetic Engineering Studies

More research efforts should be directed towards biotechnology. Studies such as plasmid curing and conjugation, development of transgenic microorganisms and transgenic crops all provide new insights to microbial control.

Better Risk Assessment of Genetically Modified Organisms (Gmos)

The commercialization of transgenic crops and transgenic microorganisms may pose a potential hazard to the ecosystem. The impact of GMOs on the surrounding environment needs to be carefully examined.

Emphasis on Sex Pheromone Research

In the future, research and development work for insect sex pheromones in Taiwan should include the following: isolation and identification of sex pheromones for key insect pests; development of methods of synthesis; improved bioassay techniques and formulations; designing effective trapping systems; evaluation of the effectiveness and economic returns of pheromone use; and extension education for farmers (Hwang 1997).

Policies Needed to Enhance Bio-Pesticide Development and Usage

The following aspects should be emphasized: strengthening the basic research; reinforcement of international and cross-strait collaboration to facilitate the commercialization; policy support and regulation improvement from the government to encourage investment on new innovations; facilitation of the use of bio-pesticides through collaboration projects among industry, academics, government officials and farmers; and promotion of the concept of organic farming to farmers.

Promotion of the Development of Novel Bio-Pesticides

Application of modern biotechnology to develop novel bio-pesticides using polyvalent Bacillus thuringiensis for plant pests control, new type of microbial protein pesticides, multifunctional symbiotic bacterium, Photor-habdus luminenscens and entomopatho-genic Beanveria bassiana, should be promoted as an endophyte with dual biocontrol activity.


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