A Study On Biological Aspects Of Oil Formulations Of Beauveria Bassiana Conidia

The entomopathogenic fungus, Beauveria bassiana, is one of the most important biocontrol agents against insect pests, and has been developed into dozens of commercial formulations in the world. This thesis sought to focus on 1) the shelf-lives of B. bassiana conidia with varying water content; 2) the suitability of different vegetable or mineral oils as carriers for formulations of B. bassiana conidia; and 3) the possible effects of the oil carriers on the shelf-lives of the conidial formulations during storage at 4 and 25℃.Impact of water content and temperature on the shelf-life of B. bassiana conidia. Powdery preparations of B. bassiana SG8702 conidia with water content (C_w) of 1.12, 4.73, 7.23, 9.84 and 14.11% were stored in dark at 4 and 25℃ during an 18-mon period and monitored at 15-day intervals for viability in order to determine their shelf-lives under controlled conditions. The C_w was found significantly affecting the viability at each temperature (P<0.05). Stored at 4℃, conidial viability always fluctuated above 91% during the first 16 months among the C_w treatments of 1.12-9.84% with no significant difference from one to another until at the end of 18 months, when the C_w treatment of 9.84% had a significantly lower viability than those with lower C_w. The viability in the high C_w treatment (14.11%) was significantly smaller than those in the C_w treatments of 1.12-9.84% from month 6 onwards and dropped to only 24.2% at the end of 12 months. Stored at 25℃, however, the viabilities differed significantly (P<0.01) among the C_w treatments of 1.12-9.84% on months 3 and 6, respectively, with a remaining viability of only 17.6% in the 9.84% treatment at the end of 6 months. The observed viabilities (V_c) depending on the (C_w) over the time of storage (T_s, months) at each temperature were well fitted to a modified survival model in the form of V_c=1/{1 + 0.0155exp[(-0.0520 + 0.0023C_w²)T_s]}at 4℃ (r²=0.859, F₄,135=409.5, P<0.0001) orV_c =1/{1 + 0.0642exp[(0.2718 + 0.0052C_w²)T_s]} at 25℃ (r² = 0.865, F₄,81=258.3, P<0.0001). When a viability of 90% was expected on a basis of the fitted model, the shelf-life of B. bassiana conidia stored at 4℃ was estimated as 5.0 months at C_w =14%, 7.3 at C_w =12%, 11.2 at C_w =10%, 14.9 at C_w =9%, 21.0 at C_w =8%, 33.0 at C_w =7%, and 65.5 at C_w=6%, respectively. Thus, the C_w for long-term storage at 4℃ could be controlled at <8%. Incontrast, for an expected viability of 80% at 25°C, the shelf-life was estimated as only 1.7 months at Cw =10%, 2.3 at Cw =8%, 3.0 at Cw =6%, 3.8 at Cw =4%, and 4.6 at Cw =2%. Obviously, an expected 3-month shelf-life required Cw <5%.Screening of oil carriers for oil formulation of B. bassiana conidia. Viscosity, biological compatibility and distributions of droplet sizes and conidia in droplets are of importance for selection of oil carriers for fungal biocontrol agents. Fourteen vegetable or mineral oil products (corn oil, soybean oil, sunflower oil, tea oil, millet oil, peanut oil, salad oil, olive oil, heavy paraffin, light paraffin, solvent #200, methylsilicone fluid, white oil #10, white oil #26) were evaluated for suitability as carriers of oil formulation of B. bassiana (109 conidia/ml) based on the above features. As a result, their viscosities varied greatly, ranging from 14.1 cSt (centistokes) in solvent #200 to 533 cSt in methylsilicone fluid. However, most of the oil formulations had a viscosity of 120-150 cSt. Sprayed under Potter Spray Tower at a consistent working pressure of 0.7 kg/cm2, the oil formulations formed droplets, of which 70-80% were <80 urn in diameter and 10-15% fell within the range of 80-120 urn. This appropriate range (40-120 urn) of droplets were most abundant in the heavy paraffin (62.1%), followed by the light paraffin (54.6%). The droplets of <40 um were most abundant in sunflower oil (44.8%) but least in the heavy paraffin (17.2% only). The percentages of the droplets of <40, 40-120 and 120-400 ^m in diameters differed significantly among the 14 oil formulations. However, significant differences from one formulation to another were not found in the counts of conidia distributed in the droplets of <40, 40-120 or 120-400 |im. The number of conidia per droplet (40-120 um) ranged from 12.9 in corn oil to 28.6 in peanut oil. The droplets of <40 um usually contained only 1 or 2 conidia per capita whereas those in diameters of 120-400 um included 200-300 eonidia per capita. Finally, paraffin, methylsilicone fluid, and white oils were well compatible to B. bassiana conidia but solvent #200, olive oil and salad oil were incompatible. With all in minds, therefore, the paraffin oils and the white oils were desirable carriers for the oil formulation of the fungal agent.Shelf-lives of B. bassiana oil formulations with single or combined oils as carriers. Twenty oil formulations of B. bassiana SG8702 conidia including heavy paraffin, light paraffin, white oil #10 and white oil #26 as carriers or combinations separately with methylsilicone fluid or peanut oil at the proportion of one seconds or thirds were examined for viabilities during long-term storage at 4 and 25°C, respectively. During the first 6 months of storage at 4°C, the viabilities examined at 15-day intervals always fluctuated above 94% in all the oil formulations except in the two combination heavy paraffin - methylsilicone fluid(3:1) and heavy paraffin - peanut oil (1:1), whose viabilities separately declined to 88.3 and 91.8% by the end of that period. Stored at 25°C, however, conidial viabilities varied largely among the oil formulations during the same period. Higher viabilities of 75.3 and 80.7% were observed separately in the combinations of white oil #26 - methylsilicone fluid (3:1) and light paraffin - methylsilicone fluid (1:1) after 6-month storage. Only were about 40-50% viabilities observed in most of the rest formulations at the same time. Unfortunately, the storage experiment that was planned to proceed for one year had to be terminated at the end of 6 months because power shortage problem in Hangzhou during summer 2004 was so serious to cause frequent interruptions of campus power supply. Such problems caused our laboratory incubators out of temperature control and may have greatly affected the viabilities of the oil formulations stored at 25°C. Therefore, no conclusion could be drawn from this part of work. Even under such uncontrolled interferences, however, the oil carriers of light paraffin and white oil #10 maintained the viabilities of 86.7 $J 89.5% 165 days after storage at 25°C, respectively. Apparently, these two mineral oils could be used as desirable single carriers for oil formulation of B. bassiana. This warrants further studies.