Effects of stabilizers on shelf-life of Epicoccum nigrum formulations and their relationship with biocontrol of postharvest brown rot by Monilinia of peaches

AIM: To find a formulation of Epicoccum nigrum conidia that maintains a high viability over time and which proves efficient to biocontrol peach rot caused by Monilinia spp. METHODS AND RESULTS: We tested the effect of stabilizers and desiccants on the shelf-life of Epicoccum nigrum conidia. Conidial samples were dried for 40 min at 40 degrees C in a fluidized bed-dryer to obtain moisture contents <15%. The toxicity of additives was tested by assaying production of conidia in fermentations and germinability of the produced conidia: 50% PEG300, 10%-5% KCl (stabilizers) and 95.24% Cl(2)Ca (desiccant) significantly (P = 0.05) reduced conidial germination. To enhance shelf-life of dried conidia, nontoxic stabilizers were added at the following different stages of the production-drying process: (i) to substrate contained in bags before production, (ii) to conidial centrifuge pellets obtained after production, before filtering and drying, (iii) to conidial centrifuge pellets obtained after production, before adding talc and drying, and (iv) to conidial centrifuge pellets obtained after production, before adding silica powder and drying. Conidial germinability was tested at 0, 180 and 365 days after storage at room temperature. Shelf-life of formulations retaining the highest viability were conidia produced with 1% KCl or 50% PEG 8000, conidia dried with 2.5% methylcellulose, and conidia dried with 1% KCl + silica powder. All these formulations improved the shelf-life of E. nigrum conidia and significantly reduced brown rot on peaches. CONCLUSIONS: Our results show that additives improve the shelf-life of E. nigrum and assist controlling brown rot on peaches. SIGNIFICANCE AND IMPACT OF THE STUDY: New improved formulations of a biocontrol agent have been obtained which will improve the control of Monilinia on peach.     

Population dynamics of Epicoccum nigrum, a biocontrol agent against brown rot in stone fruit

Aims:  To study the population dynamics of Epicoccum nigrum on peaches and nectarines and to enhance its colonization on fruit surfaces to improve its biocontrol efficacy against brown rot.
Methods and Results:  Twelve surveys were performed to study E. nigrum populations and their effect on the number of the pathogenic Monilinia spp. conidia in peach orchards in Spain and Italy between 2002 and 2005. Fresh conidia and five different formulations of E. nigrum conidia were applied three to six times to peach and nectarine trees from full flowering to harvest. The size of the E. nigrum populations was determined from the number of colony-forming units and conidial numbers per flower or fruit. Treatment with all conidial formulations increased the size of the indigenous conidial population on peach surfaces.
Conclusions:  Formulations of E. nigrum having high viability are most effective against conidia of the pathogen when applied at pit hardening and during the month immediately before fruit harvest.
Significance and Impact of the Study:  Application of an E. nigrum conidial formulation decreased the number of conidia of Monilinia spp. on fruit surfaces during the growing season to the same extent as fungicides.     

Relationship between number and type of adhesions of Penicillium oxalicum conidia to tomato roots and biological control of tomato wilt

We studied (a) the extent adhesion of Penicillium oxalicum conidia to tomato roots after application of P. oxalicum conidial formulations with or without stickers, (b) the relationship between the extent of conidial adhesion to roots and biocontrol of the conidial formulations against tomato wilt, and (c) colonisation of roots by P. oxalicum. Adhesion of P. oxalicum conidia to tomato roots occurred within the first minute of contact between the root and the conidial formulation and the bonding strength was sufficiently strong to prevent conidial removal from the roots. In addition, some formulations with stickers that increased conidial adhesion to roots improved the biocontrol of tomato wilt, when compared to that of formulations without stickers. A “dried conidia without stickers” with 0.025% Nu-Film 17 had no effect on the biocontrol of tomato wilt, despite good adherence of the conidia to the roots. The numbers of P. oxalicum conidia that adhered to the roots was constant for 60 days after application of a “dried conidia without stickers” conidial formulation. The significance of these results (speed of adhesion, number of adhered conidia, and variability of conidial external surface) are discussed in relation to the biocontrol success of tomato wilt using different types of conidial formulations with and without stickers.     

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.     

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.     

Penicillium frequentans population dynamics on peach fruits after its applications against brown rot in orchards

Aims: The study provides useful information on the temporal population dynamics of the biological control agent, Penicillium frequentans, after its applications against brown rot in orchards. Methods and Results: Population dynamics of P. frequentans were studied on peach flower and fruit surfaces after different field treatments. Eight experiments were carried out in commercial peach orchards in Spain, over four growing seasons from 2002 to 2005. Six different formulated P. frequentans conidia were applied four to six times from blossom to harvest and P. frequentans population sizes were monitored using conidial numbers and colony forming units (CFU) per flower or fruit. A consistent population of P. frequentans, ranging from 10⁵ to 10⁶ number of conidia or 10³ to 10⁴ CFU of P. frequentans per flower or fruit, was obtained. Colonization of peach surfaces by all P. frequentans formulation are similar and it appears to follow a general pattern: (i) higher colonization of fruits at preharvest than on the flowers at bloom; (ii) high populations just after treatments, especially after preharvest treatments; and (iii) a slight decline between treatments, especially in cool and moist springs. The exponential model was the most appropriate for fitting and comparing the P. frequentans dynamic populations on peaches and nectarines over time. The linearization of the P. frequentans population curve was essential to determine dynamic population and for population levels forecast. A positive relationship between number of blossom and preharvest applications, temperature, relative humidity and dynamic of P. frequentans population applied on peaches was studied using a multiple regression model. Conclusions: Blossom and preharvest applications were the most important spray times for obtaining the highest population of P. frequentans on peach surfaces. Significance and Impact of the Study: The study provides useful information on dynamic P. frequentans population and its effects on the brown rot biocontrol.     

Inhibition of Botrytis cinerea by Epirodin: A Secondary Metabolite from New Zealand Isolates of Epicoccum nigrum

Many isolates of the saprophytic fungus Epicoccum nigrum produce yellow compounds that diffuse readily into culture media. Historically, two such compounds have been identified; flavipin and epirodin both reported to have antimicrobial properties. Preliminary studies on 280 New Zealand isolates of E. nigrum confirmed that all but two produced a yellow, intensely pigmented substance in sufficient amounts to inhibit the germination of Botrytis cinerea conidia. The compound produced by the inhibitory isolates is epirodin, a polyene antibiotic. Five representative E. nigrum isolates were selected for further investigation. Two of these produced relatively large amounts of epirodin and, in a diffusible metabolite assay, reduced germination of B. cinerea conidia by up to 94%. Another isolate produced a trace amount of epirodin and had no effect on the germination of B. cinerea conidia or on germ tube morphology. The two remaining isolates produced intermediate amounts of epirodin and were only moderately inhibitory to the germination of B. cinerea conidia and to germ tube morphology. In slide dual‐culture experiments, epirodin appeared to concentrate in conidia and mycelia of B. cinerea. In acid conditions, as on dual‐culture slides of E. nigrum and B. cinerea, the yellow‐coloured epirodin underwent a hypsochromic shift, changing colour to become red. The relationship between epirodin production and the suppression of Botrytis growth and development was further investigated using necrotic kiwifruit leaf discs. The E. nigrum isolate that produced the greatest amount of epirodin almost completely inhibited the growth and development of B. cinerea on the leaf discs. In contrast, the efficacy of E. nigrum isolates which produced less epirodin ranged from 78% to just 23%. This is the first report of epirodin production by New Zealand isolates of E. nigrum, and we conclude that isolates that produce high concentrations of epirodin may have potential for plant disease control.     

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 (a_w). Conidial production was highest at high a_w(0.996) than at reduced a_w (0.98). However, conidial production at reduced a_w was improved when the a_w of the substrate was adjusted with a mixture of glycerol and water. Maximum levels ofconidiation were 7–11 × 10⁶ conidia g⁻¹ grain. The a_w 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 a_w. At reduced a_w 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 a_w, 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 a_w. A significant amount of endogenous solutes was detected in the washing liquid used for the separation of the conidia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Drying of Epicoccum nigrum conidia for obtaining a shelf-stable biological product against brown rot disease

AIMS: The effects of freeze-drying, spray-drying and fluidized bed-drying on survival of Epicoccum nigrum conidia were compared. METHODS AND RESULTS: Viability of E. nigrum conidia (estimated by measuring its germination) was 100% after fluidized bed-drying and freeze-drying, but it was determined that skimmed milk must be added in the case of freeze-drying conidia. Addition of other protectants (Tween-20, peptone, sucrose, glucose, starch and peptone + starch) to skimmed milk before freeze-drying did not improve the conidial viability which was obtained with skimmed milk alone. Glycerol had a negative effect on the lyophilization of E. nigrum conidia. Epicoccum nigrum conidia freeze-dried with skimmed milk, or fluidized bed-dried alone maintained an initial viability for 30 and 90 days, respectively, for storage at room temperature. Epicoccum nigrum conidial viability after spray-drying was lower than 10%. CONCLUSIONS: The best method to dry E. nigrum conidia was fluidized bed-drying. Conidia without protectants dried by this method had 100% viability and survived for 90 days at room temperature. SIGNIFICANCE AND IMPACT OF STUDY: This paper deals with methods for the potential formulation of a biocontrol agent which is being tested for eventual commercialization.     

Laboratory studies on the development of a conidial formulation of Esteya vermicola

The endoparasitic nematophagous fungus, Esteya vermicola, has potential as a biocontrol agent against pinewood nematode, Bursaphelenchus xylophilus. An E. vermicola conidial formulation was developed to improve conidial resistance to ultraviolet (UV), drought and heat stress. The effective concentration of each protective additive [UV protectant [fulvic acid (FA) and skim milk (SM)]; drought protectant (sorbitol) and heat protectant (calcium chloride)] was determined based on the germination rate of E. vermicola conidia after exposure to the different stressors. A combination of 0.2% FA and 4% SM, 5% sorbitol and 0.05% calcium chloride provided the most effective protection. In addition, the concentrations of spreader–sticker and antibiotic were also decided. The final formulation could be used to improve the resistance of E. vermicola conidia to multiple stressors and to increase nematode mortality compared with unformulated conidia.     

The Potential Efficacy of Glycyrrhizic Acid and Its Nanostructure Against Brown Rot of Peach fruits

Production of peaches (Prunus persica (L.) Batsch) for both local market and export is increasing each year in Egypt. Brown rot disease, caused by Monilinia laxa and Monilinia fructigena, is considered one of the most important postharvest rots affecting peaches in Egypt and economic losses are increasing. Antifungal activity of glycyrrhizic acid nanoparticles (GA-NPs) and glycyrrhizic acid (GA) at 0.2 and 0.4 mmol/L was investigated as a control for both these brown rot pathogens on peach fruits in both in vitro and in vivo studies. In the in vitro studies, GA-NPs were the most effective as shown by the ability to decrease linear growth of both brown rot pathogens in potato dextrose agar (PDA) amended with 0.4 mmol/L GA-NPs. Micrographs of M. fructigena exposed to 0.4 mmol/LGA showed mycelial deformations, nodule formation, detachment of the cell wall, shrinkage and inhomogeneous cytoplasmic materials with large vacuoles. Mycelium of M. laxa exposed to 0.4 mmol/ LGA-NPs resulted in thinner and distorted hyphae, nodule formation, cell wall thinning, and swellings. The GANPs and GA treatments improved fruit quality by maintaining firmness and total soluble solids (TSS). GA-NPs were more effective in decreasing decay incidence than their bulk material. The 0.4 mmol/L GA-NPs completely inhibited the disease on naturally infected peach fruits for both seasons of 2018 and 2019. Furthermore, 0.4 mmol/L GA-NPs reduced the disease incidence in inoculated fruits by 95 (M. laxa) and 88% (M. fructigena) in 2018 season and 96 (M. laxa) and 85% (M. fructigena) in 2019 season. In conclusion, GA-NPs could enhance the resistance of peaches against brown rot caused by M. laxa and M. fructigena.     

Formulation development of the biocontrol agent Bacillus subtilis strain CPA-8 by spray-drying

Aims: To prepare commercially acceptable formulations of Bacillus subtilis CPA‐8 by spray‐drying with long storage life and retained efficacy to control peach and nectarine brown rot caused by Monilinia spp. Methods and Results: CPA‐8 24‐h‐ and 72‐h‐old cultures were spray dried using 10% skimmed milk, 10% skimmed milk plus 10% MgSO₄, 10% MgSO₄ and 20% MgSO₄ as carriers/protectants. All carriers/protectants gave good percentages of powder recovery (28–38%) and moisture content (7–13%). CPA‐8 survival varied considerably among spray‐dried 24‐h‐ and 72‐h‐old cultures. Seventy‐two hours culture spray dried formulations showed the highest survival (28–32%) with final concentration products of 1·6–3·3 × 10⁹ CFU g^?1, while viability of 24‐h‐old formulations was lower than 1%. Spray‐dried 72‐h‐old formulations were selected to subsequent evaluation. Rehydration of cells with water provided a good recovery of CPA‐8 dried cells, similar to other complex rehydration media tested. Spray‐dried formulations stored at 4 ± 1 and 20 ± 1°C showed good shelf life during 6 months, and viability was maintained or slightly decreased by 0·2–0·3‐log. CPA‐8 formulations after 4‐ and 6 months storage were effective in controlling brown rot caused by Monilinia spp. on nectarines and peaches resulting in a 90–100% reduction in disease incidence. Conclusions: Stable and effective formulations of biocontrol agent B. subtilis CPA‐8 could be obtained by spray‐drying. Significance and Impact of the Study: New shelf‐stable and effective formulations of a biocontrol agent have been obtained by spray‐drying to control brown rot on peach.     

Optimization of storage condition for maintaining long-term viability of nematophagous fungus Esteya vermicola as biocontrol agent against pinewood nematode

The fungus, Esteya vermicola has been proposed as biocontrol agent against pine wilting disease caused by Bursaphelenchus xylophilus. In this study, we reported the effects of temperature and different additives on the viability and biocontrol efficacy of E. vermicola formulated by alginate-clay. The viability of the E. vermicola formulation was determined for six consecutive months at temperature ranged from ?70 to 25 °C. The fresh conidia without any treatment were used as control. Under the optimal storage conditions with E. vermicola alginate-clay formulation, the results suggested that E. vermicola alginate-clay formulation with a long shelf life could be a non-vacuum-packed formulation that contains 2 % sodium alginate and 5 % clay at 4 °C. Three conidial formulations prepared with additives of 15 % glycerol, 0.5 % yeast extract and 0.5 % herbal extraction, respectively significantly improved the shelf life. In addition, these tested formulations retained the same biocontrol efficacy as the fresh conidial against pinewood nematode. This study provided a tractable and low-cost method to preserve the shelf life of E. vermicola.     

Morphological characterization and optimization of conditions for conidial production of Elsinoë ampelina,the causal organism of grapevine anthracnose

Grape anthracnose, which is caused by Elsinoë ampelina, is a disease that negatively affects grape production. This study aimed to investigate the effects of aeration, temperature, light, and preculture period on the formation of E. ampelina conidia and conidial germination and virulence. The colony morphology on potato dextrose agar (PDA) plates was more diverse than that in PDA bottles. The assessment of different culture methods, temperatures, light conditions, and preculture periods revealed that optimal conidial production occurred on 25‐day‐old colonies grown in PDA bottles at 21°C for 24 hr in the dark. The cultures in PDA bottles consistently produced approximately 5.0 × 10⁶ conidia under these conditions. No conidial formation occurred when the cultures were kept at 25°C in the dark. The highest germination rate of E. ampelina was 80% at 25°C after 24 hr, whereas no germination was observed at 17°C after 12 hr. Pathogenicity tests revealed that symptoms of the disease were observed 4 days postinoculation (dpi) on leaves of Vitis vinifera cv. Red Globe. New conidia were observed on the lesions at 8 dpi. This study provides an effective method for the conidial production of E. ampelina that may also be applicable for other Elsinoë fungal species.     

Evaluation of Epicoccum nigrum for growth, morphology and production of natural colorants in liquid media and on a solid rice medium

Four nonpathogenic and nontoxigenic Epicoccum nigrum strains were evaluated for their growth, morphology and pigment producing ability in three complex and one defined liquid media. Epicoccum nigrum IBT 41028 produced pigments in all the four media tested with a maximum pigment of 3.68 AU at 410 nm in M1 medium (unoptimized) containing 5 g/l yeast autolysate. The color hue of the crude pigment extracts ranged from 74 to 102 exhibiting dark orange to green-yellow color. Pelleted morphology was shown to have a positive influence on the pigment production by E. nigrum strain IBT 41028 in the liquid media, and the use of Bis-tris buffer was found to diminish or reduce the pellet formation. Since Monascus is a well known pigment producer on rice. Pigment producing ability of E. nigrum IBT 41028 was tested on rice and compared to liquid media with Monascus ruber IBT 7904 as control. Though, both genera preferred rice but E. nigrum produced 4.6 folds higher pigment in the liquid unoptimized fermentation medium compared to M. ruber. Solid phase extraction and subsequently HPLC-DAD analysis of the crude pigment extracts showed qualitative as well as quantitative variation in the pigment composition under solid and liquid cultivations.     

Biological control of brown rot (Moniliana laxa Ehr.) on apricot (Prunus armeniaca L. cv. Hacıhaliloğlu) by Bacillus, Burkholdria, and Pseudomonas application under in vitro and in vivo conditions

In 2002 and 2003, a study was conducted to determine the effect of bacterial strains, Burkholdria OSU 7, Bacillus OSU 142, and Pseudomonas BA 8, on biological control of brown rot disease (Monilinia laxa Ehr.) on apricot cv. Hacıhaliloğlu in Malatya province of Turkey. Apricot orchard at full blooming stage was inoculated with conidial suspension (1 × 10⁶ spores/ml) of M. laxa Ehr. After inoculation, two apricot trees for each application were treated with each of the three biological control agents (Burkholdria gladii OSU 7, Bacillus subtilis OSU 142, and Pseudomonas putida BA 8) by spraying (1 × 10⁹ cfu/ml) on inoculated branches. Disease incidence was evaluated for untreated (control 1) and four different treatment groups including commercial disease management (control 2, positive control: 3% Bourdox in fall, 50% Cupper at pink flower, 30 g/100 l Corus at first blooming, and 300 g/100 l Captan at last blooming stage) and treatments including each of the three bacterial strains (OSU 7, OSU 142, and BA 8). The results showed that disease incidence for negative control (control 1) was 9.94, which was significantly higher than disease incidence for commercial application (2.57%) or bacterial treatments (2.82–5.00%) in the first year. In 2003, the lowest disease incidence observed in OSU 7 treatment (6.80%), while disease incidence rate for positive control and negative control were 9.45% and 28.46%, respectively. This result may suggest that OSU 7 has potential to be used as biopesticide for effective management of brown rot disease on apricot.     

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.This revised version was published online in October 2005 with corrections to the Cover Date.     

桃树根部内生真菌ZJ-4的分离鉴定及其对桃褐腐病的抑制效果

自桃树根部组织中分离能够防治桃褐腐病害的内生拮抗真菌,并从细胞学水平对其抑制机理进行了初步的探究。采用两点对峙法筛选抑制桃褐腐病菌Monilinia fructicola的内生拮抗真菌,通过菌落形态学特点观察及ITS基因测序分析,初步鉴定内生拮抗真菌的系统发育学地位;采用果实离体实验检测内生拮抗真菌的抗菌效果;使用电子显微镜观察被内生拮抗真菌抑制的桃褐腐病菌丝、孢子的形态以及细胞结构的变化。分离得到的内生真菌ZJ-4对桃褐腐病防效高且稳定,初步鉴定为篮状菌属Talaromyces;果实离体试验表明,内生真菌ZJ-4明显降低了桃褐腐病的发病率;通过电子显微镜观察到被内生真菌ZJ-4抑制的桃褐腐病菌丝及孢子表面粗糙凹陷,畸形现象严重,胞质溶胶收缩凝聚,细胞内部出现大量的空腔。本研究筛选的ZJ-4对桃褐腐病原菌的生长有明显的抑制作用,为开发应用该菌株提供参考。     

Persistence of <Emphasis Type="Italic">Isaria fumosorosea</Emphasis> (Hypocreales: Cordycipitaceae) SFP-198 Conidia in Corn Oil-Based Suspension

Long-term persistence of entomopathogenic fungi as biopesticides is a major requirement for successful industrialization. Corn oil carrier was superior in maintaining germination rates of Isaria fumosorosea SFP-198 conidia during exposure to 50°C for 2 h, when compared with other oils, such as soybean oil, cottonseed oil, paraffin oil, and methyl oleate. The corn oil-based conidial suspension (91.6% germination) was also better in this regard than conidial powder (28.4% germination) after 50°C for 8 h. Long-term storage stabilities of corn oil-based conidial suspension and conidial powder at 4 and 25°C for 24 months were investigated, based on the correlation of germination rate with insecticidal activity against greenhouse whiteflies, Trialeurodes vaporariorum. Viability of conidia in corn oil was more than 98.4% for up to 9 months of storage at 25°C, and followed by 23% at 21 months. However, conidial powder had only 34% viability after 3 months of storage at 25°C, after which its viability rapidly decreased. The two conidial preparations stored at 4°C had better viabilities than those at 25°C, showing the same pattern as above. These results indicate that corn oil-based conidial suspension can be used to improve conidial persistence in long-term storage and be further applied to the formulation of other thermo-susceptible biological control agents.     

Time-concentration-mortality response of potato tuber moth pupae to the biocontrol agent Cordyceps tenuipes

ABSTRACT

The potato tuber moth (PTM) Phthorimaea operculella is a critical potato pest. Larvae infest both foliage and tubers and mature larvae pupate in the soil or other safe places. Cordyceps tenuipes, an entomopathogenic fungus, infect lepidopteran pupae. To determine the effectiveness of this fungus as a biocontrol agent for PTM, we evaluated the time-concentration-mortality (TCM) response of PTM pupae to C. tenuipes using the following bioassays: (1) direct immersion in conidial suspensions, (2) incubation in sterilised or (3) unsterilised soilpremixed with conidia, and (4) incubation in unsterilised soil drenched with conidial suspensions to simulate field conditions. Fungal infection caused 100%, 83.3%, 73.3%, and 85.0% mortality of PTM pupae in assays 1–4, respectively. At 10⁸ conidia/mL or conidia/g concentration, assays 1 and 4 had short lethal times (LT₅₀) of 2.2 and 2.6 days compared with 3.7 and 4.8 days for assays 2 and 3, respectively. On day 7 after inoculation, assays 1 and 4 also had low lethal concentrations (LC₅₀) of 1.69 × 10³ conidia/mL and 1.10 × 10⁵ conidia/g compared with those of assays 2 and 3, which showed low virulence, with LC₅₀ of 3.50 × 10⁵ and 3.60 × 10⁶ conidia/mL, respectively. Our results demonstrate that C. tenuipes is a promising candidate for PTM biocontrol at the pupal stage. Drenching the soil surface with conidial suspensions may be the most effective method of field application.