S. Pascual

Surface hydrophobicity, viability and efficacy in biological control of Penicillium oxalicum spores produced in aerial and submerged culture

The surface hydrophobicity, viability and biocontrol ability of Penicillium oxalicum spores, produced either in aerial or submerged culture, were characterized. A phase distribution test showed that spores produced in both methods of culture were highly hydrophobic, but those produced in aerial culture were more hydrophobic. Spores stored fresh at either 4 or 25 °C retained a high viability (80%) after 27 weeks of storage, although aerial spores survived better. Freeze‐drying severely affected viability, especially of submerged spores. Biocontrol ability against Fusarium oxysporum f. sp. lycopersici was studied in the growth chamber. Aerially‐ produced spores were more effective than submerged ones. Aerially‐produced P. oxalicum spores appeared to have more advantages than those produced by submerged culture, in relation to both viability and efficacy. These results demonstrate that physiological changes occur depending on production conditions which significantly influences quality of the biocontrol agent.

Ecophysiological factors affecting growth, sporulation and survival of the biocontrol agent Penicillium oxalicum.

The effect of temperature, pH, water potential and sources of nitrogen and carbon on the biocontrol agent Penicillium oxalicum were studied in vitro. The fungus is xerotolerant, mesophillic and has a wide pH tolerance. The parameters evaluated (germination, germ tube length, growth rate and sporulation) showed different sensitivities to the environmental factors. Peptone and free amino acids gave the highest growth rates and high levels of sporulation. Xylose, mannose and fructose gave the highest growth rates and mannose induced strong sporulation. The effect of nutrients (mannose + arginine) and water potential was also studied in vivo. The xerotolerant character of the fungus was confirmed. From this study we consider Penicillium oxalicum ecologically competent to perform effectively as a biocontrol agent in the soil environment.

Water availability affects the growth, accumulation of compatible solutes and the viability of the biocontrol agent Epicoccum nigrum

Growth of the biocontrol fungus Epicoccum nigrum was more sensitive to ionic solute water stress (NaCl) than non-ionic (glycerol) on potato dextrose-based media at −0.5, −3.0 and −5.5 MPa water potentials. Subsequent physiological manipulation of growth of E. nigrum in glycerol-modified media to −3.0 MPa water potential resulted in a significant increase in the accumulation of compatible solutes in both mycelial liquid cultures and spores, but no enhanced accumulation of the desiccation protectant trehalose, when compared to unmodified media (−0.5MPa). The main solute accumulated was glycerol, followed by arabitol. In temporal studies over 20 days maximum accumulation of glycerol occurred in 5-d old cultures with water stressed cultures having 250× greater amounts than those from unmodified medium. The arabitol content was also higher in mycelium and spores produced under water stress. The difference was maximum after 15 days growth. Glucose content decreased over time in mycelial colonies but increased in spores. The germination of conidia from the two treatments was similar, regardless of compatible solute content, even at −9.25 MPa water potential stress. However, germ tube extension was significantly increased at this water potential level. The production of E. nigrum spores at −3.0 MPa water potential resulted in improved survival when stored fresh at 4 and 25 °C. However, freeze-drying severely affected the viability of spores produced on both media (−0.5 or 3.0 MPa). Accumulation of compatible solutes may assist the fungus in better ecological competence and establishment in the phyllosphere, where water availability is often limited.

Accumulation of compatible solutes in Penicillium frequentans grown at reduced water activity and biocontrol of Monilinia laxa.

The biocontrol fungus Penicillium frequentans was grown on potato dextrose broth of either - 0.4 MPa water potential (unstressed) or reduced water potential (stressed) media modified with glycerol to - 7.0 MPa. The endogenous compatible solutes and sugars were quantified after 5, 10 and 15 days growth. Glycerol was the main compatible solute accumulated at reduced water potential, with a concomitant decrease in erythritol and mannitol, and the sugar, glucose. Maximum accumulation of glycerol occurred in 5-day-old cultures, with the content in P. frequentans biomass produced at reduced water potential being about 180 times higher than that produced in unmodified medium (- 0.4 MPa). There was no enhanced accumulation of the desiccation protectant trehalose. Homogenisation of cultures, for spray applications of the fungus, resulted in a partial loss of the solutes accumulated in the fungal biomass. P. frequentans controlled peach brown rot ( Monilinia laxa ) on peach twigs, with the disease index being significantly smaller when treated with the antagonist produced at reduced water potential. However, both inoculum types were able to reduce the length of lesions to a similar extent. This suggests that the inoculum of the antagonist produced in medium with freely available water (- 0.4 MPa) becomes rapidly adapted to the dry environment in the phyllosphere, counteracting the possible initial advantage of the inoculum produced at reduced water potential (- 7.0 MPa).

Production of the fungal biocontrol agent Epicoccum nigrum by solid substrate fermentation: effect of water activity on accumulation of compatible solutes

Epicoccum nigrum conidia were produced by solid fermentation on wheat grains (cv. Rendeveaux and Brigadier) at different water activities (aw). Conidial production was highest at high aw(0.996) than at reduced aw (0.98). However, conidial production at reduced aw was improved when the aw of the substrate was adjusted with a mixture of glycerol and water. Maximum levels ofconidiation were 7–11 × 106 conidia g−1 grain. The aw of the solid substrate affected the pattern of accumulation of compatible solutes in the conidia. Mannitol was the main polyol in all conidialtypes. However, the amounts of mannitol were higher in conidia produced at high aw. At reduced aw the conidia of E. nigrum accumulated moreglycerol, which is more efficient in the osmorregulation proccess than mannitol. Arabitol accumulated in low amounts, specifically in conidia produced at the lower aw, on cv. Rendeveaux but not on cv. Brigadier. Trehalose was detected in higher amounts in cv. Rendeveaux than in cv. Brigadier, andthe amounts were higher in conidia produced at high aw. A significant amount of endogenous solutes was detected in the washing liquid used for the separation of the conidia.

In vitro studies on the effects of fungicides on beneficial fungi of peach twig mycoflora

In vitro studies were carried out to investigate a possible integrated use of chemical and biological means to control the peach twig blight pathogen,Monilinia laxa. Three fungal antagonists ofM. laxa (Penicillium purpurogenum, Penicillium frequentans andEpicoccum nigrum) and six fungicides (vinclozolin, iprodione, thiram, captan, benomyl and thiophanate-methyl) were used in the study. Sensitivity of the fungal isolates to the fungicides was determined in vitro by calculating ED50 values. Benomyl and thiophanate-methyl were the most fungitoxic compounds and captan was the least fungitoxic.M. laxa andP. purpurogenum were the most sensitive to all chemicals tested, whileE. nigrum andP. frequentans presented bigger differences in their sensitivity to chemicals compared toM. laxa. E. nigrum was consistently less sensitive to benomyl (ED50=2.26 ppm), thiophanate-methyl (ED50=9.61 ppm) and vinclozolin (ED50=3.89 ppm) than the other fungi.P. frequentans was less sensitive to captan, vinclozolin, iprodione, thiophanate-methyl and thiram thanM. laxa (8, 7, 5, 4 and 2 times respectively). These results suggest thatE. nigrum andP. frequentans could be successfully used in an integrated control programme that combines biological and chemical methods.

Nutritional requirements of antagonists to peach twig blight,Monilinia laxa, in relation to biocontrol

Different carbon and nitrogen sources and accessory substances were tested to determine their effect on the growth and sporulation of the peach twig blight pathogen,Monilinia laxa, and of three of its antagonists (Penicillium frequentans, Penicillium purpurogenum andEpicoccum nigrum), since the success in twig blight biological control by treatments with the fungal antagonists depends on the type of nutrients added to the antagonist formulation. Combinations of sucrose-ammonium tartrate, glucose-(NH4)3PO4-folic acid and lactose-KNO3 were selected from these laboratory experiments because they enhanced the growth and sporulation ofP. frequentans, P. purpurogenum andE. nigrum, respectively, but notM. laxa. In glasshouse experiments, twig blight was reduced following the application of mixtures of antagonists with the corresponding enhancing nutrients.