Study On Biological Characteristics Of Paecilomyces Lilacinus(Thom.)Samson Str.NH-PL-03 And Its Biological Effects Of Secondary Metabolites

Studies on the culturing characteristics of Paecilomyces lilacinus have been carried out. The growing indexes including OD value, pH value, dry weight of mycelia, sporulation and the substrate concentration(C,N) of Paecilomyces lilacinus (Thom) Samson str. NH-PL-03 in the liquid culturing process had been systemically investigated. The principal components of growing indexes had been analysed and the liquid culturing stage had been cluster analyzed. The results showed that: 1) The rhythm growing were interrelated to C and N consuming significantly, 2) Dry weight of mycelia, sporulation and OD (620nm) value were the principal components of nutritious growth, reproduction and physiological status respectively, 3) Based on the cluster analysis, the cultural process can be divided into four stages: The first one (1² d) was an adapted stage, all growing indexes gradual changing; The second one (3⁵d) was a growth stage, all growing indexes gain in rapidly ,biomass increase significantly; The third stage (6⁷d) was a logarithmic stage, all growing indexes leaping forward and the biomass increased rapidly; The fourth one (after 8 d) was a stagnant stage, all growing index was steady relatively.The system investigated on the HPLC analysis . POD Isoenzyme and P-glucosidase activity, solvable protein of fresh mycelia in the process of liquid culturing have been carried out, the main results were summarized as follows:Analysed the soluble protein from the mycelia of Paecilomyces lalicinus in the duration of liquid culturing process with SDA-PAGE, the result showed that: there were common bands in the all culturing duration. At the early duration, more of bands were produced, at the middle term, the bands decreased, but to the end of culturing duration, the bands increased. That could be deduced that the presence of soluble protein bands was a good indicator of culturing process.The POD isoenzyme from the mycelia of Paecilomyces lalicinus in the process of liquid culturing has been electrophoresis analyzed. The result showed that: there were four affinity isoenzymetic bands produced on the gel. The Rf. value were 0.098, 0.137, 0.294 and 0.373 respectively. In the general trend, the bands number decreased along with the liquid culture. In the 3⁵th day of liquid culturing, there were three bands produced on the gel. In the 6th day, two bands produced, but to the lately duration (from 7¹0^th)only one band existed, this result suggested that the patterns of isoenzyme was a sign of culturing duration.All culturing periods could produce cell-wall-degrading enzymes such as p-glucosidase, at the early term (1^6th) the activity of P-glucosidase increased gradually, from 7.0u/mL to 9.438u/mL. In the 7^th day of culturing duration the activity grew up abruptly, from 9.438 u/mL to 43.875 u/mL, at 8^th day reach 55.0 u/mL,as time goes on, the activity maintain dynamic balance.Studies on culturing characteristic (effect of different media, aerating condition, initial pH setting, temperature, C,N setting ) on growth of Paecilomyces lilacinus .have been carried out.The OD value, pH value, dry weight of mycelia, sporulation in the process of culture has been investigated to study the growing characteristics of Paecilomyces lilacinus on different media. Result showed that Paecilomyces lilacinus could grow normally on all tested media Best mycelia biomass occurred on the media of PDA, Czapek and Martin & Johnson, cultured for 8d. the dry weight of mycelia could reach 0.80g/50mL, 0.73g/50mL and 0.70g/50mL respectively; The max. sporulation of 36.90x 106/mL and 35.51x106/mL occurred on Czapek and oat media respectively.The different aerating condition was determined by shaking speed setting, the result showed that: shaking speed is one of a key factor for Paecilomyces lilacinus growth. It is found that lOOr/min of shaking speed was the suitable aerating condition for mycelia biomass and sporulation, under this aerating condition , the dry weight of mycelia reached 1.10g/50mL at 8d, sporulation was 28.5><106/mL. Next one was 180r/min setting, the sporulation could reach 23.12xlO6/mL.A series of experiments was conducted to investigate the effect of different aerating condition on growth of Paecilomyces lilacinus. The different aerating condition was determined by setting different liquid loading. The result showed that: 50mL/250mL of liquid loading was the best aerating condition for mycelia biomass and sporulation. under this aerating condition , the dry weight of mycelia reached 0.83g/50mL and sporulation was 23.50x 106/mL at 8d.The growing index including OD value (620nm) , pH value, dry weight of mycelia, spore number, dynamic of C,N concentration in the process of liquid culture under different initial pH setting have been investigated. Result showed that Paecilomyces lilacinus exhibited growth at pH 4.4-8.5, meanwhile had varying growth and sporulation responses to different initial pH, optimum mycelia growth occurred at pH 5.0⁷.5, and for sporulation at pH 5.0-6.5, the max. dry weight of mycelia of 1.08g/50mL could reach with initial pH of 5.0 at lOd and spore number reach peak value of 23.66xlO6/mL at 6.5 of initial pH. pH value of 2.3 and 9.5 could showed obvious inhibited effect on growth of Paecilomyces lilacinus.Studied on effect of temperature on Paecilomyces lilacinus growth, had been carried out. The growing index (OD value ,pH value, dry weight of mycelia, spore number) at the different temperature (15-35"C) had been investigated. The result showed that: temperature is key factor to culturing, Paecilomyces lilacinus exhibited normal growth in 20³0°C, Between in 15³0°C scope, higher temperature could speedy the C,N consuming. The optimum mycelia growth and sporulation occurred at25 °C, mycelia biomass could reach 1.08g/50mL at 9d. spore number reach 40.12* 106/mL at 10th day of culturing.The growing index ( OD value.pH value, dry weight of mycelia, spore number) in the different Carbon Sources and Nitrogen source formulation had been investigated, the result showed that: Paecilomyces lilacinus could grow on all tested formulation, glucose benefited for mycelia growth, sugar was more suitable for sporulation. Organic M such as soybean powder and fish powder was better for sporulation than inorganic N (NH4 or NO3) .Generally speaking, simple carbon and nitrogen resource formulation was suitable for mycelia growing, organ carbon and nitrogen formulation was better for sporulation. Cultured with the formulation (glucose and peptone) , the dry weight of mycelia increased along with the culturing, it could reach 1.05g/50mL at 7d. The max. sporulation of 28.99* 106/mL could reach at 8d cultured with second formulation contain with sugar and peptone, next one was the fourth (glucose and soybean powder) , the sporulation was 20.13*106/mLat 8d.Paecilomyces lilacinus can grow normally on all tested media with different C/N rate (varied from 1 to 10) ,and the different C/N of media can make different in culturing parameter .generally speaking: C/N sitting in 2³ (C/N=3, 3.3 * 2) was suitable to biomass developing., the dry weight of mycelia increase along with the culturing. it could reached 1.12g/50mL> 1.18g/50mL and 0.78g/50mL respectively at 9do but lower and higher C/N sitting (10 or 1) was good for sporulation, the sporulation could reach 21.23* 106/mL at 6d and 18.56* 106 -IVmL at 9d respectively.The effect of different substrate concentration on growth of Paecilomyces lilacinus was studied, the growing index (OD value, pH value, dry weight of mycelia, spore number), dynamic of substrate concentration in the process of liquid culture under different substrate concentration have been investigated. Result showed that: substrate concentration could make different in culturing parameters. To some degree, lower substrate concentration ( C+N=0.5g/100mL) was good for sporulation increasing quickly, substrate concentration setting of O.lg/lOOmL, the spore number reach peak value 12.21 xlO6 at 5d ,the middle substrate concentration setting (C+N=0.5g/100mL> 1.5g/100mL)benefit for biomass, the max. mycelia biomass was 1.18g/50mL and 1.08g/50mL at 9¹0d. The highest substrate concentration (C+N=13.5g/100mL) carried inhibited effect on growth of Paecilomyces lilacinusStudied on the effects of nine kinds of pesticide on the conidial germination and colonies growth of Paecilomyces lilacinus have been carried out The result showed: all tested pesticide could inhibit the conidial germination and colonies growth in its degree. CuSO4, Thiophanate-methyl, Fenpyroimat and Parathion-methyl had significant inhibitd both on conidial germination and colonies growth. Fenvalerateand Dimehypo were weak relatively, and that of Omethoate, dichlorros and Trichlorphon ranked intermediately.Paecilomyces lilacinus cultured on different medium, temperature, and aerating condition and its efficacy against juveniles of Ditylenchus destrnctor Thorne had been investigated. The result showed that: as a whole ,the NH-PL-03 filtrate has significant toxic effects on Ditylenchus destrnctor Thorne, the revised mortality of treated juveniles increase along with culture continues. Culturing medium rich in Cn N source and mineral ion (such as Czapek) expressed higher toxic effects (43.4%) . 30"Cof culture temperature and lower aerating condition (60r/min of rotating speed and 40% of loading capacity) exhibited higher nematicidal activity, in this culturing condition, the max. revised mortality of Ditylenchus destrnctor was 48.8%, 47.1% and 57.1% respectively.The studies on physiological activities of metabolites from Paecilomyces lilacinus (Thorn.) Samson str. NH-PL-03 has carried out. The secondary metabolites coming from inner-cell and inter-cell, effect of the crude preparation on the mug bean sprouts have been investigated, it caused suppress or increases results on sprouts (length of bud) , obviously suppressed at high concentration, significantly increased at low concentration. The biological activities of inner-cell crude preparation was more significant than the inter-cell, the crude preparation from inner-cell inhibited the bud length by 64.62%, meanwhile the inter-cell only by 31.92%. The crude preparation cultured on Martin and Johnson media exhibited more activities than other media; it increased sprouts by 54.58% at concentration (1:16) . By and large, activities increased along with culture time; the best culture time was 8¹2d. The inner-cell preparation could remain certain activities treated by heat. But both of suppress effects and elongation effects (1:32) decreased by 25.86% and 7.96%.Studies were conducted to assess the inhibition effect of the secondary metabolite from Paecilomyces lilacinus on Fusarium oxysporum Schl. Tests result showed that the secondary metabolite in different concentration, different culturing term and cultured by different media would express different inhibiting results on the spore germination and colony growth of Fusarium oxysporum. Judged by appearances, the inhibited colony grew slowly and badly-distributed on the media. Studied on effect of different concentration on the inhibition results had been carried out. The inhibited rates increased along with the concentration, treated by 5% secondary metabolite after 7d, the inhibition rates were 80.56%, but treated with concentration of 1%, the inhibition rates were 42.78%.The inhibition results of the antagonistic secondary metabolite cultured at a constant term of 1-1 Id had been investigated. Tests result showed that: treated with antagonistic secondary metabolite cultured at a constant term of 4¹0d, the relativeinhibiting rates of all treatment were above 70% at 4% concentration after 7d. But the relative inhibiting rates gone down by 48.49% when treated with 1 lth days secondary metabolite.The secondary metabolite cultured by different media had varying antagonistic results against Fusarium oxysporum. When treated with secondary metabolite cultured by Czapek, PDA and Martin-Johnson media, the highest inhibiting rate to colony and sporulation were over 72% and. 90% respectively, meanwhile the data tested by LSD, result showed that there were no inhibition differences were found between the antagonistic material cultured on Czapek media and Hymexazol (*400 ) after 7d. It concluded that Paecilomyces Hlacinus strain NH^PL⁰3 was promising used as biological agent against Fusarium oxysporum.Studies were conducted to assess the antagonistic effect and mechanism of Paecilomyces Hlacinus against Fusarium oxysporum Schl. Tests result showed that the secondary metabolite produced by Paecilomyces Hlacinus obviously suppressed the growth of the colony and spore germination of Fusarium oxysporum Schl. The antagonistic mechanism against Fusarium oxysporum Schl mainly to produce cell-wall-degrading enzymes such as p-glucosidase and antibiotic material (such as antibiotic protein and amylose ) and nutrient competitive action between the Paecilomyces Hlacinus and Fusarium oxysporum Schl.This paper reported the experiments of phoxim-degradation by Paecilomyces lalicinus under liquid culturing. When mix cultured Paecilomyces lalicinus and phoxim (40ug/mL ) for 5d, the properties were quickly dissipated along with liquid culturing, the degrade rate could reach 100%- with acetone and (1/1) as extraction solvent ,under 40°C and HOr/min condition .shaking for 30min, the known added recovery rates of phoxim were ranged from 98.85% to 90.12%, and the average value was 93.42%.,When mix cultured Paecilomyces lalicinus and phoxim (400ug/mL ) for 5d, the properties were quickly dissipated along with liquid culturing, the degrade rate could reach 96%¹00%? Effect of different properties concentration on the degrade results also was investigated. The degrade rates decreased along with the concentration increase, at the concentration of 8000ug / mL after 5d, the degrade rates were 25.51%, but at 400ug / mL, the degrade rates were 98.78%. Judged by appearances, the colony grew slowly and badly-distributed on the media contented with phoxim at 400 or 800|ig/mL. Culture on different media, Paecilomyces lalicinus express different degrade efficacy to phoxim. When it cultured on Czapek, PDA and Martin-Johnson media, the highest degrade rate to phoxim were over 98%. Studies were conducted to assess the effect of constant subculture on the degrade result, tests result showed that: there were no differences were found between the all treats of subculture, mix cultured...