Preparation of dry flowable formulations of Clonostachys rosea by spray drying and application for Sclerotinia sclerotiorum control

WU Hong-qu, SUN Li-li, LlU Fang, WANG Zhi-ying CAO Chuan-wang

1 College of Forestry, Northeast Forestry University, Harbin 150040, P.R.China

2 Hubei Biopesticide Engineering Research Centre, Wuhan 430064, P.R.China

1.lntroduction

Clonostachys rosea (syn.Gliocladium catenulatum) is a mycoparasite capable of antagonizing a range of plant pathogens, such as Alternaria spp., Bipolaris sorokiniana,Botrytis cinerea (Yu and Sutton 1997; Jensen et al.2002,2004), Fusarium culmorum (Huang 1978; Knudsen et al.1995), Rhizoctonia solani (McQuilken et al.2001),Sclerotinia sclerotiorum (Rodríguez et al.2011), Pythium spp.(Chatterton and Punja 2009), and Verticillium dahlia(Keinath et al.1991).The biological control mechanisms of C.rosea are similar to those of Trichoderma spp.including nutrient competition, mycoparasitism, induced resistance of the plants, antimicrobial metabolites, detoxification of mycotoxins and antibiotics (Lorito et al.2010; Druzhinina et al.2011; Mukherjee et al.2012).Certain Trichoderma and Clonostachys species can also promote plant growth and elicit induced resistance that can protect plants from pathogen attack (Hermosa et al.2012; Mukherjee et al.2013).C.rosea has been used worldwide as a biological control agent (Jensen et al.2007; Vargas et al.2009).Recent studies demonstrated that C.rosea and its fermentation broth can inhibit mycelial growth and conidia germination of many plant pathogens (Ma et al.2004).Pot trials revealed that C.rosea not only effectively controls diseases but also promotes plant growth (Wang et al.2001; Wang et al.2015).Some studies indicated C.rosea is efficacious for controlling nematode damage (Dong et al.2004).

Dry flowable formulation is a relatively new type of formulations and is being developed as safely and commercially attractive alternatives to wettable powder and suspension concentrate (Walters et al.2014).Spray drying is widely used to produce dry flowable formulations and is being evaluated as an alternative to freeze-drying for the stabilization of biotherapeutics.Dry flowable formulations contain inert ingredients that prolong the shelf life and increase the efficacy of the products.The present study aimed to produce a C.rosea dry flowable formulation from spores and their fermentation broth.The resulting C.rosea dry flowable formulation is a green bio-fungicide with great commercial prospects and advantages such as long shelf life, good disintegration, high suspensibility,fine particle size, and greater resistance to rain wash-off.This study also investigated the effects of the C.rosea dry flowable formulation on the control of S.sclerotiorum under Petri dish and pot conditions.S.sclerotiorum (Lib.)de Bary is a global soil-borne plant pathogen (Bolton et al.2006).S.sclerotiorum attacks more than 500 plant species, including many economically important crops, such as oilseed rape/canola (Brassica spp.), alfalfa (Medicago sativa L.) (Purdy 1979), and soybean (causing Sclerotinia stem rot), resulting in significant yield losses (Grau and Hartman 1999).The findings of our study will help improve the practical application of C.rosea for bio-control of major plant pathogens.

2.Materials and methods

2.1.Fungal strain and pathogens

C.rosea strain WY-1 (1010conidias g-1) was isolated and deposited in the Lab of Forest Microbiology, Northeast Forestry University, China.The pathogen S.sclerotiorum was collected from infected rape plants and deposited in our laboratory.Potato dextrose agar (PDA) medium (200 g of potato, 20 g of dextrose, 18 g of agar, and 1 000 mL of distilled water) was used as the standard growth and storage medium for S.sclerotiorum.

2.2.lnert ingredients

The inert ingredients, including wetting agents, dispersants,UV protection agents, adhesives, disintegrants, and the fumed silica, attapulgite clay, celatom, kaoline, and bentonite carriers used in this study were obtained from the National Pesticide Engineering Research Center, Nankai University,China.The ingredients were classified as follows: wetting agents (Morwet EFW, K12); dispersants (lignin sulfonic acid calcium, Morwet D425, NNO); UV protection agents (nano zinc oxide); adhesives (CMC-Na, sodium ligninsulfonate,PEG, oxalic acid, and dextrin); and disintegrants (povidone K30, (NH4)2SO4, croscarmellose sodium, crospovidone, and carbamide).

2.3.Effect of fungal antagonists on S.sclerotiorum growth

The C.rosea and S.sclerotiorum mycelial discs with a diameter of 4 mm were inoculated in PDA medium in a culture plate (9 cm diameter).S.sclerotiorum was inoculated after growth of C.rosea for 2 days.The two inoculation positions were 1.5 cm away from the plate edge and symmetrical with respect to the diameter line of the culture plate.The interaction between C.rosea and S.sclerotiorum was observed after 4 days of culture in an incubator at (28±1)°C.C.rosea and S.sclerotiorum were also inoculated separately in PDA medium in culture plates as controls.The inhibition ratio was calculated using the following formula: Inhibition ratio (%)=(Diameter of control-Diameter of treatment)/(Diameter of control-4 mm)×100, where, 4 mm is the diameter of the incubation strain mycelial discs.

2.4.Formulations

C.rosea fermentation broth preparationAfter the reserved C.rosea strain WY-1 was cultured at 28°C for 120 h,the mycelial discs of C.rosea strain WY-1 were removed with a 5-mm-diameter puncher and incubated in 300 mL of PD medium (200 g of potato, 20 g of dextrose and 1 000 mL of distilled water) in 500-mL flask at 180 r min-1at 28°C for 10 days.The sterile fermentation broth of C.rosea was obtained after the fermentation broth filtering 0.22-μm membrane to remove conidia and mycelium.

Biocompatibility screening of inert ingredientsCarriers(500 mg mL-1), wetting agents (120 μg mL-1), dispersants(250 μg mL-1), disintegrants (500 μg mL-1) and adhesives(250 μg mL-1) were added to PDA medium plates.A total of 0.1 mL aliquot of conidia solutions (103conidia mL-1) was uniformly spread on the mixed PDA medium containing inert ingredient in each plate, and the PDA medium without inert ingredient was used as a control.Each medium was treated with conidia solution for four replicates and stored in a culture incubator at (28±1)°C for 72 h.

The mycelium growth rate was measured according to the method of Weitz et al.(2001) with modifications.Briefly, a 10-mm mycelial discs was cut from actively growing colonies in the central PDA plates containing each inert ingredient using a 10-mm-diameter puncher.A 10-mm mycelial discs as the control was cut from actively growing colonies in the central PDA plates without inert ingredient.Each treatment was performed in four replicates, and the diameter of the mycelium colonies was measured every 24 h during 7 days.The mycelium growth rate was calculated using the following formula: Growth rate (mm d-1)=Diameter of mycelium colony(mm)/Number of growth days (d)

Orthogonal optimization of the formulationFor the orthogonal design, the ratios of disintegrants and adhesives were determined on the basis of the particle size and wetting time.For spray drying, the inlet-air temperature (factor A), air-outlet temperature (factor B), atomization pressure(factor C) and feed flow rate (factor D) were optimized based on their effects on the conidial viability of C.rosea.Optimum disintegrants and adhesives, and spray drying conditions were selected based on the four-factor, three-level orthogonal experiments (Appendices A and B).The wetting agents, dispersants and UV protective agents were selected based on previous tests as follows (Shao et al.2014): 1%EFW, 2% K12, 7% D425, 7% NNO, and 0.3% xanthan gum.The methods of inert ingredient screening are as follows.

(i) Wettability: A representative sample of powder((5.0±0.1) g) on a Petri plate was dropped on the surface of water in a beaker.A stop-watch was started, and the time(to the nearest second) required for complete wetting was recorded.The experiment was repeated five times, and the results were averaged.

(ii) Disintegrating property: A sample of granules (0.5 g,particle size: 250-1 414 μm) was added to a 100-mL measuring cylinder containing 90 mL of distilled water at 25°C.The measuring cylinder was sealed with a stopper and rolled at a speed of 8 r min-1until the sample was completely disintegrated.A disintegration time of less than 3 min was considered acceptable.

(iii) Conidial viability of C.rosea: A total of 1 mL aliquot of C.rosea conidial suspension (106conidias mL-1) was incubated on PD plates at 180 r min-1at 28°C.After a 20-h incubation, conidia on mycelial discs were suspended into water.Conidial concentrations were quantified in a hemocytometer and calculated as the conidial number per square centimeter of colony.

2.5.Homogenization and spray drying of the C.rosea formulations

The selected inert ingredients were mixed uniformly with universal pulverizer (YS-04A, Beijing Yanshan Masanori Machinery Co., Ltd., China) and mixed superfinely with an appropriate amount of water using a high-pressure homogenizer (AH100B, 500-1200 bar, Canada).After the treated inert ingredients and the fermentation broth were homogenized, the dry flowable formulations of C.rosea were produced by spray drying.The dry flowable formulations of conidia powder and freeze-dried powder of the C.rosea fermentation broth as the ratio of 1:3, 1:1 and 3:1 were formulated by spray drying and named after I, II and III, respectively.The single dry flowable formulation of conidia power and freeze-dried powder of the C.rosea fermentation broth were formulated as control by spray drying, respectively.

2.6.Physical characteristics and storage stability of the C.rosea formulations

The physical characteristics of the formulations, including suspensibility, wettability, pH, dispersibility, disintegratability,particle size and moisture content, were analyzed according to the Collaborative International Pesticides Analytical Council (CIPAC) standards.Microbial insecticides are usually sensitive to high temperature and should be stored at cool temperature.C.rosea conidia easily lose viability under high-temperature condition.Thus, the viability of the C.rosea conidia in the dry flowable formulation was evaluated by testing its stability at room temperature for 24 mon.A 90-g sample of the C.rosea dry flowable formulation was stored in a 200-mL sealed coex plastic bottle at room temperature.Each treatment was repeated three times.

2.7.Pot trial

The pathogen was cultured in PD liquid medium for 6 days after activation in a PDA plate for 3 days.Inoculation(105conidias mL-1) was performed by spraying on cucumber seedlings (two-leaf age) maintained under humid condition until sclerotinia rot was observed.The light transmittance of S.sclerotiorum was 3.6%.The C.rosea dry flowable formulation was sprayed to control sclerotinia rot at dilutions of 200×, 400×, 800×, 1 600× and 3 200×.The control efficacy was observed after 15 days, and the sclerotinia rot disease index was recorded in accordance with the Grading Criteria for Disease index (1-9 grades; Appendix C).

Disease index=∑(Disease grading×Number of leaves at this scale)×100/(9×Total number of leaves investigated)

Control efficacy (%)=(Disease index of untreated control-Disease index of treatment)/Disease index of untreated control×100

2.8.Data analysis

All statistical analyses were performed using SPSS 18.0(SPSS Inc., Chicago, IL, USA).Differences between treatments were assessed by one-way analysis of variance(ANOVA) followed by Student-Newman-Keuls multiplerange tests at 0.05 level.

3.Results

3.1.Effects of the fungal antagonist on the growth of S.sclerotiorum in co-culture

The plate antagonism test indicated that C.rosea inhibits S.sclerotiorum, which simultaneously inhibits C.rosea.The inhibition ratio of C.rosea against S.sclerotiorum was(57.07±2.07)%.The relative antagonism ratio was 2.22±0.27.The co-culture of C.rosea and S.sclerotiorum demonstrated that C.rosea can effectively inhibit sclerotium formation,indicating that C.rosea can control this pathogen (Fig.1).

3.2.Biocompatibility screening of inert ingredients

The wetting agents and dispersants were screened based on biocompatibility tests with C.rosea according to Shao et al.(2014).The effects of different disintegrants and adhesives on the viability of C.rosea are presented in Tables 1 and 2.

The effects of five disintegrants on C.rosea conidias are presented in Table 1.Compared to the control, only carbamide and crospovidone significantly decreased conidia viability, and (NH4)2SO4significantly promoted mycelium growth.However, povidone K30 and croscarmellose sodium did not significantly affect C.rosea conidias.Crospovidone and carbamide significantly inhibited mycelium growth compared to the control.Based on the effects of the disintegrants on conidia viability and mycelium growth,povidone K30, (NH4)2SO4and croscarmellose sodium were employed as disintegrants for C.rosea dry flowable formulations.

Fig.1 Co-culture of Clonostachys rosea and Sclerotinia sclerotiorum for 4 days at 28°C on potato dextrose agar.A, C.rosea.B,co-culture of C.rosea and S.sclerotiorum.C, S.sclerotiorum.

Table 1 Effects of five disintegrants on Clonostachys rosea conidia

Table 2 Effects of five adhesives on Clonostachys rosea conidia

The effects of five adhesives on C.rosea conidias are presented in Table 2.Compared to the control, CMCNa, sodium ligninsulfonate and PEG, did not significantly affect the viability of conidias, whereas oxalic acid and dextrin slightly decreased the viability of conidias.The five adhesives did not significantly affect mycelium growth,although PEG slightly increased mycelium growth.For the adhesive screening, conidia viability was considered the major factor because conidias played a key role in pathogen control.Therefore, CMC-Na, sodium ligninsulfonate, PEG and oxalic acid were selected as adhesives for C.rosea dry flowable formulations.

3.3.Spray drying parameters

The effects of four factors (air-inlet temperature, factor A;air-outlet temperature, factor B; atomization pressure, factor C; feed flow rate, factor D) on the viability of C.rosea were compared by orthogonal experiment.The optimized spraydrying process parameters (A3B2C3D2) were an air-inlet temperature of 140°C, an air-outlet temperature of 60°C, an atomization pressure of 2.5 MPa, and a feed flow rate of 600 mL h-1(Appendix B).Analysis of variance indicated that the effects of the four factors on spray drying decreased in the following order: atomization pressure＞feed flow rate＞airinlet temperature＞air-outlet temperature (Appendices D and E).

3.4.Orthogonal optimization of the formulation

Screening of wetting agents and dispersantsThe inert wetting agents and dispersants were selected based on previous results (Shao et al.2014).The four-factor,three-level orthogonal test indicated that the optimal ratio of these inert ingredients was A2B3C3D3, corresponding to 3% Morwet EFW, 4% K12, 10% Morwet D425, and 9%NNO (Appendix F).Analysis of variance indicated that the effects of the four factors on the suspension rate of the dry flowable formulation decreased in the following order:D＞B＞C＞A (Appendices F and G).

Screening of disintegrants and adhesivesFirstly, 4%disintegrants and 3% adhesives were screened based on conidia viability and were then mixed with wetting agent,dispersant agent, C.rosea conidia powder and carrier up to 100% to produce C.rosea dry flowable formulations.The disintegration time and particle size of the C.rosea dry flowable formulations were determined.Finally, the disintegrants and adhesives were analyzed by a two-factor,three-level orthogonal test based on disintegration time and particle strength.The best group of these inert ingredients was A3B3C1D1, corresponding to 5% croscarmellose sodium, 5% (NH4)2SO4, 0.5% CMC-Na, and 1% oxalic acid.Analysis of variance ranked the effects of these factors as C＞D＞B＞A.The best ratio of components in the C.rosea dry flowable formulation was 30% C.rosea (conidias and fermentation broth), 3% Morwet EFW, 4% K12, 10% Morwet D425, 9% NNO, 5% croscarmellose sodium, 5% (NH4)2SO4,0.5% CMC-Na, 1% oxalic acid and carrier palygorskite up to 100% (Appendices H-J).

3.5.Physical characteristics of the dry flowable formulation

A dry flowable formulation of C.rosea was successfully developed by orthogonal optimization.This dry flowable formulation contained the following inert ingredients: Morwet EFW, K12, Morwet D-425, NNO, CMC-Na, croscarmellose sodium, (NH4)2SO4, CMC-Na and oxalic acid.The physical properties of the formulation, as determined according to CIPAC standards, were as follows: conidia viability≥80%,moisture≤1.63%, weight suspensibility≥78.29%, spore suspensibility≥82.12%, wettability≤19 s, pH (6.5±0.2) and an average particle size of 10.84 μm.

3.6.Storage stability

At 3, 6, 12, and 24 mon, test samples of the C.rosea dry flowable formulation were collected from the sealed coex plastic bottles at room temperature, and the conidia density of the dry flowable formulation was analyzed (Table 3).The conidia loss was no greater than 20% after 24 mon.

Table 3 Physical characteristics of dry flowable formulation

3.7.Pot trials

The control effect of the C.rosea dry flowable formulations on S.sclerotiorum is presented in Table 4.An obvious synergetic effect of the C.rosea conidia powder and its fermentation broth in the dry flowable formulation was observed.The highest inhibitory ratio among mixed formulations were in descreasing order of mixed formulation I (83.33%)＞mixed formulation III (79.92%)＞mixed formulation II (78.67%).The inhibitory effects of all three mixed formulations were greatly superior to those of the single formulation.For mixed formulation I,the inhibitory rate of 200× mixed formulation I was the highest (83.33%) among all concentrations, and the inhibitory rates of all concentrations were significant except for the 400× and 800× concentrations.The inhibitory rate of the C.rosea fermentation broth dry flowable formulation (I-2) was higher than that of the C.rosea conidia powder dry flowable formulation (I-1) at all concentrations.The inhibitory rate of mixture II ranged from 54.17 to 78.67%, and the differences between the inhibitory rates were significant at all concentrations except for 400× and 800×.The control efficiency of the C.rosea fermentation broth dry flowable formulation (II-2) was higher that of the C.rosea conidia powder dry flowable formulation (II-1).The inhibitory rate of mixture III ranged from 51.67 to 79.92%, and the control efficiency of the C.rosea conidia powder dry flowable formulation (III-3) was higher than that of the C.rosea fermentation broth dry flowable formulation (III-1) by 10.91-14.34% at identical concentrations.The differences in the inhibitory rates of the two single preparations were significant(P＜0.05).

4.Discussion

As an alternative to chemical pesticides, C.rosea has exhibited remarkable potential for biocontrol because of its good biological characteristics, great ability for environmental adaptation and wide parasitism (Sutton et al.1997).C.rosea can also be mixed with other biocontrol agents to control plant diseases and pests.For example, Phytophthora palmivora on coca were successfully controlled by a mixture of C.rosea and Trichoderma spp.in Peru, broadening the host range and improving environmental adaptation of C.rosea (Krauss et al.2013).C.rosea (B10+P8) mixed with Bacillus thuringiensis to control a gastro-intestinal nematode in sheep was more effective than either C.rosea or B.thuringiensis alone (Baloyi et al.2012).Beauveria bassiana conidia powder was formulated with C.rosea conidia powder as a carrier and delivered by bees on tomatoes and sweet peppers in cages in the greenhouse.This mixture of B.bassiana and C.rosea not only controlled the whitefly and tarnished plant bug, with 49 and 73% of mortality, respectively, but also inhibited gray mold on tomato flowers and leaves by 57 and 46%, respectively, and on sweet pepper flower an leaves by 59 and 47%, respectively (Kapongo et al.2008).However,the usage of a mixture of C.rosea conidia powder and its fermentation broth as a biocontrol agent has not been reported.

Recently, spray drying has been widely applied into drying conidia of pest pathogen fungi and disease antagonism (Larena et al.2003; Horaczek et al.2004; Guijarro et al.2006).However, the spray drying process severely damages the conidia, resulting in low viability.For example, spray drying did not succeed to protect several fungal conidia, including conidia of T.harzianum(Larena et al.2003; Guijarro et al.2006).Conversely, microbial activity was

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protected by formulation processing using spray drying technology.This spray drying technique was successfully applied for the microencapsulation and dry flowable granular production of bio-insecticides such as Lactobacillus plantarum (Rajam and Anandharamakrishnan 2015), Trichoderma harzianum(Munoz-Celaya et al.2012), B.thuringiensis (Zhou et al.2008), B.subtilis B99-2 (Ma et al.2015), and B.subtilis strain T429 (Meng et al.2015).In the present study, the C.rosea conidia powder and its fermentation broth were mixed to produce the C.rosea dry flowable formulation using spray drying technology.This dry flowable formulation of C.rosea exhibited increased longevity and stability of the C.rosea conidia and enriched the properties of formulation of C.rosea.The plate antagonism test indicated that the C.rosea dry flowable formulation was very effective against S.sclerotiorum, and the pot trial subsequently demonstrated that the inhibition of S.sclerotiorum by the C.rosea dry flowable formulation reached 83.33%.The dry flowable formulation of C.rosea was composed of 30% C.rosea(ratio of conidia powder and its fermentation broth is 1:3),3% Morwet EFW, 4% K12, 10% Morwet D425, 9% NNO,5% croscarmellose sodium, 5% (NH4)2SO4, 0.5% CMC-Na,1% oxalic acid and palygorskite (carrier) up to 100%.The C.rosea conidia powder and its fermentation broth exhibited synergetic effects when mixed to produce the dry flowable formulation.

5.Conclusion

In summary, the dry flowable formulation of C.rosea described here overcomes the slow efficacy of bioinsecticides and low persistence of chemicals.The C.rosea dry flowable formulation is a promising green bioinsecticide with long shelf life, quick disintegration, high suspensibility, small particles, strong adhesion and rainfastness.A dry flowable formulation with small particles and, consequently, a broad surface area can be obtained by the spray-drying production process.The broad surface area of small particles facilitates interactions with microorganisms and essential nutrients for growth, and thus small particles are preferred for the reactivation of encapsulated microorganisms (Doherty et al.2011).Our study provides a new practical and theoretical basis for the further development and application of C.rosea as a biocontrol agent.

Acknowledgements

This work was supported by grants from the Fundamental Research Funds for the Central Universities, China(2572016DA02), the National Natural Science Foundation of China (31570642) and the Science and Technology Innovation and Entrepreneurship Projects of Returned Overseas Personnel in Jilin Province, China (2013-36).In addition, we thank Associate Professor Kou Junjie, National Pesticide Engineering Research Center, Nankai University,China, for providing inert ingredients.

Appendicesassociated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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