Journal of Plant Pathology (2017), 99 (2), 391-401 Edizioni ETS Pisa, 2017 391

FIELD APPLICATION OF SCLEROTIAL MYCOPARASITES AS BIOCONTROL AGENTS TO CEPIVORA, THE CAUSE OF WHITE ROT

I.E. Elshahawy, N.M. Saied, F. Abd-El-Kareem and A.A. Morsy

Plant Pathology Department, Agricultural and Biological Research Division, National Research Centre, Giza, Egypt

SUMMARY INTRODUCTION

Three fungal isolates from a total of 30 were selected Onion (Allium cepa L.) is one of the most economically on the basis of their antagonistic activity against the most important vegetable crops grown in Egypt. The total culti- pathogenic isolate (Sc2) of Stromatinia cepivora (Berk.) vated area was 66452 ha and yielded 2,438,436 metric tons Wetzel, the causal agent of onion white rot. These isolates in 2014/2015 growing season (Anonymous, 2015). White were identified as Chaetomium globosum (Chg6) Kunze, rot, caused by Stromatinia cepivora (Berk.) Whetzel, is a Clonostachys rosea (Cr12)(Link) Schroers and Penicillium disease restricted to Allium spp. under field conditions, and oxalicum (Po9) Currie & Thom. In vitro, the selected iso- is widespread in many parts of the world (Abd-Elrazik et lates were seen to colonize the surface of sclerotia and al., 1973; Hunger et al., 2002; Metcalf et al., 2004; Ulacio- cause collapse to their outer rind. Parasitized sclerotia Osorio et al., 2006; Sallam et al., 2009; El-Sheshtawi et al., appeared degraded and failed to germinate when they 2009; Elsherbiny et al., 2015; Elshahawy et al., 2017). The were transferred onto fresh potato dextrose agar plates pathogen persists in soil as a dormant, spherical, small (0.3- indicating that the sclerotia of S. cepivora had been killed 0.6 mm in diameter), black sclerotia which can survive for by these mycoparasites. Also, cultural filtrates of these several years in the absence of host plants (Coley-Smith et isolates inhibited the growth of S. cepivora (Sc2) and de- al., 1987). Sclerotia are the primary source of inoculum. creased germination of sclerotia. Control of onion white Allium-specific root exudates, called alkyl cysteine sul- rot disease by soil treatment with the inoculum of these phoxides, leach out of Allium plant roots and biodegrade sclerotial mycoparasites was attempted. Under greenhouse in soil to form volatile alkyl disulphides (Coley-Smith and and field conditions, all sclerotial mycoparasites signifi- King, 1969). These volatiles stimulate S. cepivora sclerotia to cantly reduced the incidence and severity of onion white germinate (Coley-Smith and King, 1969), and produce hy- rot disease. P. oxalicum (Po9) was the most effective with phae which grow through the soil in search of host tissue. the lowest percentages of white rot incidence and severity. Once contact is made with Allium plant root, the hyphae Ch. globosum (Chg6) and C. rosea (Cr12) occupied the sec- form an appressorium, and then produce an infection plug, ond rank in this respect. Also, these treatments increased which penetrates between the cell wall junctions and deep vegetative growth of live onion plants in greenhouse and into the root. Infected plants also exude more alkyl cyste- increased onion bulb yield in field compared with control. ine sulphoxides, stimulating increased sclerotial germina- Mycoparasites enhanced biocontrol activity of peroxidase, tion in the soil surrounding them (Coley-Smith and King, polyphenoloxidase and chitinase enzymes in onion plants 1969). The hard-walled sclerotia formed during infection grown in field to resist infection with S. cepivora. can remain dormant yet viable in the soil for many years, meaning that once a field is infested with S. cepivora, Al- Keywords: sclerotial mycoparasites, biocontrol, onion lium cultivation may not be possible there for many years white rot, Stromatinia cepivora. (Coley-Smith et al., 1989). No field treatment has yet been developed to completely eradicate the from soil. There are few effective chemicals or other methods to control onion white rot (Banks and Edgington, 1989), and host resistance is not sufficient to provide commercially acceptable control (Earnshaw et al., 2000). As a result, at- tempts to manage the disease have focused on reducing the populations of sclerotia in the soil. Therefore, the objec- tives of the present study were: (i) to monitor potential of mycoparasites occurring in sclerotia of S. cepivora, (ii) to select the most efficient mycoparasites against the most ag- gressive Stromatinia strain to further greenhouse and field Corresponding author: I.E. Elshahawy Fax: + 20 57 2403868 studies, (iii) to study the indirect effects (cultural filtrate) E-mail: [email protected] apart from parasitism against S. cepivora, (iv) to study the 392 Sclerotial mycoparasites as biocontrol agents Journal of Plant Pathology (2017), 99 (2), 391-401 efficacy of three selected mycoparasites in reducing scle- The individual plants were rated for disease severity with rotial survival and Stromatinia injuries in greenhouse and a scale of 0 = healthy plants, 1 = slightly severe (yellowing natural field conditions and (v) to study the activation of of the leaves, reduced root system), 2 = moderately severe the defense mechanisms induced by mycoparasites in onion (yellowing and die-back of leaves, root system badly de- host in field conditions. cayed), 3 = severe (complete yellowing of the plant, die- back of the leaves, semiwatery soft rot of scales and roots) and 4 = highly severe (completely dead plants, extensively MATERIALS AND METHODS decayed bulbs and roots). Disease severity was calculat- ed according to the formula described by Zewide et al. Location of the experiments. This study was carried (2007) with some modification: Disease severity = ∑ (Dis- out at the Plant Pathology Department, Agriculture and ease scale × Number of plants in each scale)/Total number Biological Division, National Research Centre as well as of plants × Highest disease scale. Percent data of disease within fields of El-Qalubia governorate Egypt. These fields incidence were statistically analyzed after arcsine square were highly infested with S. cepivora and characterized by root transformation; however, untransformed data are high sclerotial density (Elshahawy et al., 2017). presented. For the analysis of ordinal data such as disease severity, nonparametric analysis was used based on the Stromatinia cepivora isolates. A wet-sieving floatation ranks of the data, but untransformed data are presented. technique that facilitates the rapid isolation of sclerotia The data were analysed with SPSS software version 14.0. of S. cepivora from highly infested soil sites in El-Qalubia Analysis of variance was determined and the mean values governorate, Egypt was used (Utkhede and Rahe, 1979). were compared by Duncan’s multiple range test at P < 0.05. The sclerotia were surface disinfected, placed onto potato The isolate with the highest pathogenicity of 10 isolates dextrose agar (PDA) plates and incubated at 18°C to ob- investigated, isolate Sc2, was selected and used throughout tain pure cultures. the present study. Ten isolates of S. cepivora were Pathogenicity test. Sclerotia ob- subjected to pathogenicity testing using onion seedlings. Isolation of sclerotial mycoparasites. tained from infested soil were rinsed in sterile distilled The experiment was carried out in pots under greenhouse water; surface sterilized in 0.5% sodium hypochlorite for conditions (the minimum and the maximum temperature 2 min and transferred to PDA plates. After incubation for ranges were 5-10°C and 20-25°C, respectively) using the susceptible cultivar, Giza red, according to the method 7 days at 18 ± 2°C, fungi growing from ungerminated scle- reported in a previous study (El-Sheshtawi et al., 2009). rotia were subcultured on fresh PDA plates. All cultures The experiment was carried out in autoclaved (121oC for were maintained on PDA at 18 ± 2°C for further study. 2 h) clay soil in sterilized plastic pots (30 cm diameter, 4 kg each). Inoculum of S. cepivora isolates was obtained by Testing the antagonistic behavior of isolated fungi growing each isolate on PDA plates at 18°C for 60 days. against S. cepivora. Each of fungi isolated was tested for A suspension of sclerotia was obtained by adding 100 ml antagonism to S. cepivora (Sc2) using the dual culture tech- sterile distilled water to each plate and then blending for niques (Bell et al., 1982). PDA plates were inoculated on 20 s at low speed. After filtration, the sclerotia were trans- one side with a 5 mm mycelial disk from a 7-day-old cul- ferred onto filter paper and counted under a microscope ture of the test fungus. The opposite side was inoculated with fine forceps. Inoculum of each S. cepivora isolate with a disc of S. cepivora and the plates were incubated at was placed on the top of the soil surface at a rate of 1 ml 18 ± 2°C in the dark. Four replicate plates were made for sterile distilled water containing 100 sclerotia/kg soil ac- each test fungus. After 14 days of inoculations, the follow- cording to the method of Metcalf et al. (2004) and then ing parameters were measured: (1) average growth area of covered with additional soil to fill pots to 5 kg each. A S. cepivora (Sc2) and the percentages of growth reduction, set of pots drenched with the same amount of water free and (2) average number of formed sclerotia per cm2 and from fungal sclerotia served as control. Five healthy onion the average inhibition zone. The classification groups were transplants (60 days old) were transplanted into each pot adapted from Ghaffer (1969). Data of the growth area, after immersion in 0.5% sodium hypochlorite solution number of formed sclerotia and zone of inhibition were for 3 min and washing several times in sterilized distilled subjected to SPSS software version 14.0 and analyzed sta- water. Four pots of each isolate were used as replicates. tistically by the analysis of variance test (ANOVA); the dif- Nitrogen fertilizer in form of urea (46% N) was added ferent means were compared by Duncan’s multiple range at the rate of 10 g/pot 30 days after planting and plants test at P < 0.05. Based on their antagonistic efficacy, three were irrigated when necessary. The percentages of disease of the fungal antagonists were subjected to identification incidence were calculated after 100 days after transplant- tests according to the methods described (Gilman, 1957; ing as follows: Disease incidence = No. of infected plants/ Barnett and Hunter, 1972; Watanabe, 1994; Domsch et No. of total plants × 100 (Brix and Zinkernagel, 1992). al., 1980; Ahammed et al., 2005). The three isolates were Journal of Plant Pathology (2017), 99 (2), 391-401 Elshahawy et al. 393 identified as Clonostachys rosea (Link) Schroers, Chaeto- of germinating sclerotia were determined. Percent data of mium globosum Kunze and Penicillium oxalicum Currie germinated sclerotia were statistically analyzed after arcsine & Thom. square root transformation; however, untransformed data are presented. The data were subjected to SPSS software Parasitism of sclerotia. Sclerotia of S. cepivora (Sc2), version 14.0. Analysis of variance was determined and the produced in culture by the method of Coley-Smith (1959), mean values were compared by Duncan’s multiple range were surface sterilized in 1% sodium hypochlorite for test at P < 0.05. 4 min, rinsed in sterile distilled water for 5 min and placed around the periphery of 7-day-old colonies of the test fungi Inoculum of mycoparasites. Sterilized (121°C for grown on water agar plates. Plates were sealed to prevent 60 min) wheat bran was used for preparation inoculum desiccation and incubated at 18 ± 2°C. After 3 weeks, of mycoparasites C. rosea, Ch. globosum and P. oxalicum sclerotia were examined microscopically for evidence of (Mahdizadehnaraghi et al., 2015). Carboxymethyl cellulose parasitism. Uninfected sclerotia and sclerotia naturally (1%) was used as an adhesive at the rate of 1:10 (v/w). The infected with C. rosea, Ch. globosum and P. oxalicum were pH was adjusted to neutral by adding CaCO3 at the rate also examined microscopically. A sample from each group of 15 g/kg. The mixture was sterilized and then mannitol of parasitized sclerotia was removed and transferred to was added as osmoticant at the rate of 8.5 ml of 3% manni- fresh PDA to determine their viability. tol per 100 g formulation. Subsequently, each fungal spore suspension (106 spores/ml) was individually incorporated Testing the antagonistic behavior of cultural filtrate of into sterilized wheat bran carrier under aseptic conditions sclerotial mycoparasites. Cultural filtrates of C. rosea, Ch. at the rate of 50 ml of suspension per 100g and thoroughly globosum and P. oxalicum were tested for antagonism for mixed with a sterilized spoon. The materials (35% mois- S. cepivora growth and sclerotia germination using PDA ture content) were packed in polythene bags, sealed and plates amended with cultural filtrates of each of sclerotial stored at room temperature. mycoparasites. Potato dextrose broth (PDB) medium (1 l containing 200 g potato and 20 g dextrose, pH 6.5-6.8) was Greenhouse experiments. The greenhouse experi- prepared and 100 ml was distributed into 250 ml sterile ments were conducted with a completely randomized de- flasks. A 5 mm mycelial disk from a 7-day-old culture of sign (CRD) with 5 treatments (3 mycoparasites C. rosea, the test fungus was added to each flask. Three replicate Ch. globosum and P. oxalicum, infected control, and healthy flasks were prepared. The flasks were incubated on a rotary control) each with four experimental units. Experimental shaker at room temperature for 10 days. The cultures were unit consisted of a sterilized plastic pot (30 cm diameter) then filtered off under vacuum and the filtrate centrifuged containing 5 kg of autoclaved onion field soil pre-infested at 2500 g for 20 min. Twenty-five ml of the supernatant were with S. cepivora (Sc2) sclerotia at the rate of 100 sclerotia/ re-filtered through a sterile 0.22 mm Millipore filter direct- kg soil according to the method of Metcalf et al. (2004) ly into 225 ml molten PDA. The amended PDA was poured as previously described. The inoculum of mycoparasite into Petri dishes and after cooling, the plates were centrally fungi was added (at the rate of 1% w/w) to the infested inoculated with a 5 mm mycelial disc from the edge of a soil one week before onion transplanting. Five surface dis- 7-day-old colony of S. cepivora. Unamended PDA medium, infected 60-days-old onion transplants (cv. Giza red) were prepared as above, was used as control. Five replicate plates transplanted into each pot. The experiment was carried of each filtrate were made. The plates were incubated at out during the 2014/2015 growing season and repeated in 18 ± 2°C for 14 days. Colony diameter was recorded and the the 2015/2016 growing season. Onion was planted in De- means were compared by Duncan’s multiple range test at cember to evaluate disease control in April. The minimum P < 0.05 with SPSS software version 14.0. Qualitative analy- and the maximum temperature ranges were 5-10°C and 20- sis of any changes in growth form was recorded visually. (−) 25°C, respectively. Nitrogen fertilizer in the form of urea means no change, (+) means colonies compact and dense, (46% N) was added at the rate of 10 g/pot 30 days after hyphae more branched and no sclerotia formed and (++) planting; the plants were irrigated when necessary. Disease means no growth. On the other hand, sclerotia of 60-day- incidence and severity of onion white rot was recorded as old cultures of S. cepivora (Sc2) were collected from the previously described. At the end of the experiment (100 edges of Petri plates, then soaked in test tubes containing days after transplanting), plant height (cm), number of the filtrate to be tested for 12 h at room temperature. At the leaves/plant and biomass (g) of alive plants were mea- end of the dipping period, sclerotia were washed with ster- sured. Data collected from greenhouse experiments were ile distilled water and thirty sclerotia from each treatment analyzed separately for each growing season. Percent data were transferred individually under aseptic conditions to of disease incidence were statistically analyzed after arcsine Petri-dishes containing PDA medium. Water-soaked sclero- square root transformation; however, untransformed data tia were used for control treatment. Four replicates (dishes) are presented. For the analysis of ordinal data such as dis- were used for each treatment. Petri dishes bearing sclero- ease severity, nonparametric analysis was used based on the tia were incubated at 18 ± 2°C for 7 days and percentages ranks of the data, but untransformed data are presented. 394 Sclerotial mycoparasites as biocontrol agents Journal of Plant Pathology (2017), 99 (2), 391-401

Table 1. Pathogenicity test of ten Stromatinia cepivora isolates harvest (150 days after transplanting) as follows: Disease against onion transplants (cv. Giza red). incidence = No. of infected plants/No. of total plants × 100 (Brix and Zinkernagel, 1992). Root systems of the harvest- (a) (b) S. cepivora isolate Disease incidence (%) Disease severity ed plants were examined. White rot severity was evalu- (c) Sc1 80.4 ± 4.24 e 0.8 ± 0.19 bc ated on a scale of 0 to 6, where 0 = 0%, 1 = (1 to 14%), Sc2 95.6 ± 0.97 a 1.0 ± 0.00 a Sc3 91.1 ± 0.82 b 0.7 ± 0.16 c 2 = (15 to 35%), 3 = (36 to 64%), 4 = (65 to 85%), 5 = (86 to Sc4 91.4 ± 0.71 b 0.8 ± 0.16 bc 99%), and 6 = 100% of the root system decayed. Disease Sc5 90.2 ± 1.79 b 0.8 ± 0.16 bc severity was calculated according to the formula described Sc6 88.3 ± 1.63 bc 0.9 ± 0.16 ab by Zewide et al. (2007) with some modification: Disease Sc7 85.2 ± 4.00 cd 0.7 ± 0.16 c Sc8 84.1 ± 1.63 d 0.8 ± 0.28 bc severity = ∑ (Disease scale × Number of plants in each Sc9 84.4 ± 1.46 d 0.7 ± 0.28 c scale)/Total number of plants × Highest disease scale. At Sc10 83.2 ± 2.44 de 0.9 ± 0.16 ab the end of the experiment, onion plants (bulbs with the Negative control 0.0 tops of the plants) within each plot were weighed. Data (a) The percentages of disease incidence were calculated after 100 days collected from field experiments were analyzed separately after transplanting as follows: Disease incidence = No. of infected plants/ No. of total plants × 100 (Brix and Zinkernagel, 1992). Values are means for each experimental site. Percent data of disease inci- of 4 replicates with 5 onion seedlings each. dence were statistically analyzed after arcsine square root (b) The individual plants were rated for disease severity with a scale of transformation; however, untransformed data are present- 0 = healthy plants, 1 = slightly severe (yellowing of the leaves, reduced root ed. For the analysis of ordinal data such as disease sever- system), 2 = moderately severe (yellowing and die-back of leaves, root sys- tem badly decayed), 3 = severe (complete yellowing of the plant, die-back ity, nonparametric analysis was used based on the ranks of of the leaves, semiwatery soft rot of scales and roots) and 4 = highly severe the data, but untransformed data are presented. The data (completely dead plants, extensively decayed bulbs and roots). Disease se- were subjected to SPSS software version 14.0. Analysis of verity was calculated according to the formula described by Zewide et al. variance was determined and the mean values were com- (2007) with some modification: Disease severity = ∑ (Disease scale × Num- ber of plants in each scale)/Total number of plants × Highest disease scale. pared by Duncan’s multiple range test at P < 0.05. Values are means of four replicates with 5 onion seedlings each. (c) Means ± standard deviations within a column followed by the same Determination of enzymes activities. The effect of in- letter are not significantly different by Duncan multiple range test at oculum of mycoparasites added to the soil on the activities P < 0.05. Arcsine square root transformed data for disease incidence (%) and the nonparametric rank test for disease severity were used for statistic of the defense enzymes of peroxidase, polyphenoloxidase analysis; however, untransformed data are presented. and chitinase of onion plants was estimated at 60 days after planting. To extract the enzyme, onion leaf samples (g) were homogenized with 0.2 M Tris HCl buffer (pH 7.8) The data were analyzed with SPSS software version 14.0. Analysis of variance was determined and the mean values at 0°C containing 14 mM β-mercaptoethanol at the rate of were compared by Duncan’s multiple range test at P < 0.05. 1/3 w/v. The extracts were obtained by filtering off the debris with a clean cloth and centrifuging at 3,000 rpm for 15 min. The supernatants were recovered and kept in Field experiments. Naturally infested soil with S. ce- pivora located at El-Deer region, El-Qalubia governorate a tube in an ice bath until assayed. The supernatant was was chosen for estimating the efficiency of soil treatment used to determine the activity of enzymes by using a UV with inoculum of mycoparasites for controlling white rot spectrophotometer. Peroxidase activity was assayed with disease of onion plants under field conditions. The ex- guaiacol as the hydrogen donor as described by Ham- periments were conducted with a completely randomized merschmidt et al. (1984) and expressed as the increase in design (CRD) with 4 treatments (3 mycoparasites C. rosea, absorbance at 470 nm/g fresh weight/minute according to Ch. globosum and P. oxalicum, and a non-amended con- the method described by Lee (1973). Polyphenoloxidase trol), each with four experimental units. An experimen- enzyme activity was determined by measuring the rate of tal unit consisted of 3 rows. The dimensions of each row quinone formation as a result of oxidizing 3,4-dihydroxy- were 6 m in length, 30 cm in height and 50 cm in width. phenylalanine (DOPA) and polyphenoloxidase activity The inoculum of mycoparasites, freshly prepared, was in- was expressed as the increase in absorbance at 475 nm/g corporated into the soil at the rate of 300 g formulation/m fresh weight/min according to the method described by length of the row. Therefore, a cavity of 15 cm in depth Bashan et al. (1985). The determination of chitinase en- was made in the surface of each row. The inoculum of zyme was carried out using colloidal chitin as substrate mycoparasites was added to this cavity and then recov- and dinitrosalicylic acid (DNS) as reagent to measure ered with the soil and immediately irrigated. One week reducing sugars according to the method described by after incorporation, ninety surface disinfected 60-days- Monreal and Reese (1969). Chitinase activity was ex- old onion transplants (cv. Giza red) were transplanted in pressed as mM N-acetylglucosamine equivalent released/ each row. Irrigation and fertilization were conducted as gram fresh weight/60 min at 540 nm. Data of enzymatic generally recommended for onion production regimes. activity were subjected to SPSS software version 14.0 and The percentages of disease incidence were calculated at analyzed statistically by the test ANOVA and the different Journal of Plant Pathology (2017), 99 (2), 391-401 Elshahawy et al. 395

Table 2. Interaction between three isolates of mycoparasites and Stromatinia cepivora in dual culture at 14 days after inoculation.

Mycoparasite (a) Reaction type (b) Average growth area (cm2) Average inhibition zone (cm2) Average No. of sclerotia (cm2) (c) Parasitism Ch. globosum (Chg6) B (d) 28.75 ± 1.91 d 0.00 ± 0.00 c 40.0 ± 3.26 b + C. rosea (Cr12) D 44.00 ± 1.63 b 5.75 ± 1.91 b 41.0 ± 2.82 b + P. oxalicum (Po9) D 40.50 ± 2.00 c 8.75 ± 1.91 a 40.5 ± 1.15 b + Control − 70.00 ± 0.00 a 0.00 ± 0.00 c 80.0 ± 0.00 a − (a) Reaction type produced in dual culture. B, the growing margins of the two colonies meet, S. cepivora is inhibited and overgrown by the other fungus; D, the growth of S. cepivora is inhibited at a distance, leaving a clear zone of inhibition between the two organisms. (b) Growth area (cm2) of S. cepivora was calculated using the planimeter. (c) (−) unparasitized, (+) sclerotia parasitized. (d) Means ± standard deviations within a column followed by the same letter are not significantly different by Duncan multiple range test at P < 0.05. means were compared by Duncan’s multiple range test Parasitism of sclerotia. The isolates of Ch. globosum at P < 0.05. (Chg6), C. rosea (Cr12) and P. oxalicum (Po9) were seen to colonize the surface of sclerotia of S. cepivora (Table 2). The inner tissues of sclerotia appeared degraded and resulted RESULTS AND DISCUSSION in collapse of the outer rind. Parasitized sclerotia failed to germinate when they were transferred onto fresh PDA The pathogen. Sclerotia of S. cepivora collected from plates, indicating that the sclerotia of S. cepivora had been highly infested soil sites in El-Qalubia governorate, Egypt, killed by these mycoparasites. proved to belong to ten isolates of S. cepivora (Table 1). Pathogenicity test of these isolates in onion transplants cv. Effect of cultural filtrates of mycoparasites on S. ce- Giza red (Fig. 1) showed that they exhibited varied degrees pivora growth. Cultural filtrates of the tested mycopara- in severity. Isolate Sc2 was the most aggressive one and sites had significant effect on the growth of S. cepivora recorded the highest disease incidence (95.0%) and disease (Table 3), showing a fungistatic effect toward S. cepivora, severity (1.0). Isolate Sc1 recorded the lowest percentage and also causing substantial reduction in both linear of disease incidence (80.4%) and moderate aggressiveness growth and changes in growth form. P. oxalicum (Po9) (0.8 severity), while isolates Sc3, Sc7 and Sc9 recorded was the most effective followed by Ch. globosum (Chg6) the lowest disease severity (0.7). Due to its aggressiveness and C. rosea (Cr12). Seven and 14 days after inoculation, toward onion transplants, Sc2 isolate was selected as the the growth of S. cepivora in the presence of filtrates of main isolate for further trials. Ch. globosum (Chg6), C. rosea (Cr12) and P. oxalicum

Detection of antagonistic activity. A total of 30 fun- LEGENDS TO THE FIGURES gal isolates were isolated from sclerotia of S. cepivora and tested for antagonistic activity using the dual culture tech- a nique. The predominant genera found to colonize scle- 120 95.6 91.1 91.4 90.2 100 88.3 85.2 rotia were Trichoderma spp., Penicillium spp., Fusarium 80.4 84.1 84.4 83.2 spp., Chaetomium spp. and Clonostachys spp. Three iso- 80 lates were chosen for further investigations on the bases 60 of quantitative analysis of their ability to inhibit growth 40 20 of S. cepivora and qualitative analysis of their ability to Disease incidence (%) 0 damage the sclerotia of S. cepivora. Characteristics of the Sc1 Sc2 Sc3 Sc4 Sc5 Sc6 Sc7 Sc8 Sc9 Sc10 interaction between these isolates and S. cepivora are given Stromatinia cepivora isolates in Table 2. Chaetomium globosum isolate (Chg6) produced type B reaction inhibiting the growth of S. cepivora and b growing over the colony. Clonostachys rosea (Cr12) and 1.2 1.0 Penicillium oxalicum (Po9) isolates displayed type D reac- 1 0.9 0.9 2 0.8 0.8 0.8 0.8 tion, producing zones of inhibition of 5.75 and 8.75 cm , 0.8 0.7 0.7 0.7 respectively. The inhibition in the growth and number of 0.6 sclerotia formed by S. cepivora caused by Ch. globosum 0.4 Disease severity (Chg6), C. rosea (Cr12) and P. oxalicum (Po9) were 58.9, 0.2 0 50.0%, 37.1, 48.8% and 42.1, 49.4%, respectively. P. oxa- Sc1 Sc2 Sc3 Sc4 Sc5 Sc6 Sc7 Sc8 Sc9 Sc10 licum (Po9) was the most effective, followed by C. rosea Stromatinia cepivora isolates (Cr12) and Ch. globosum (Chg6). No significant differenc- es were found between isolates in inhibition of sclerotia Fig. 1. Disease incidence (a) and severity (b) of ten Stromatinia formation. Fig.cepivora 1. Disease (Sc)incidence isolates (a) and againstseverity (b) onion of ten Stromatinia transplants cepivora (cv. (Sc) Giza isolates red). against onion transplants (Giza red cv.). 396 Sclerotial mycoparasites as biocontrol agents Journal of Plant Pathology (2017), 99 (2), 391-401

Table 3. Growth of S. cepivora in the presence of 10% cultural Table 4. Germination (%) of sclerotia of S. cepivora soaked in filtrates of sclerotial mycoparasites in vitro. cultural filtrates of sclerotial mycoparasites.

S. cepivora growth (mm) (a) Change in Mycoparasite Germinations (%) Reduction (%) Mycoparasite After 7 days After 14 days growth form Ch. globosum (Chg6) (a) 42.5 ± 3.30 c 56.0 C. rosea (Cr12) 47.5 ± 6.40 b 50.9 Ch. globosum (Chg6) (b) 11.3 ± 3.00 c 16.0 ± 3.26 c + C. rosea (Cr12) 17.0 ± 2.30 b 19.0 ± 2.30 b + P. oxalicum (Po9) 28.9 ± 3.11 d 70.1 P. oxalicum (Po9) 08.5 ± 1.15 d 12.5 ± 5.77 d + Control 96.7 ± 5.47 a − Control 90.0 ± 0.00 a 90.0 ± 0.00 a − (a) Means ± standard deviations within a column followed by the same letter are not significantly different by Duncan multiple range test at (a) (−) no change; (+) colony compact and dense, hyphae more branched P < 0.05. Arcsine square root transformed data for sclerotia germination and no sclerotia formed. (%) were conducted for statistic analysis; however, untransformed data (b) Means ± standard deviations within a column followed by the same are presented. letter are not significantly different by Duncan multiple range test at P < 0.05.

Table 5. White rot incidence (%) and severity in soil artificially infested with S. cepivora and amended with sclerotial mycopara- sites in greenhouse during two growing seasons.

(a) White rot incidence (%) and severity Mycoparasite 2014/2015 growing season 2015/2016 growing season Incidence (%) Severity Incidence (%) Severity Ch. globosum (Chg6) (b) 30.0 ± 23.09 b 0.2 ± 0.11 b 30.0 ± 23.09 b 0.2 ± 0.12 bc C. rosea (Cr12) 35.0 ± 38.29 b 0.3 ± 0.37 b 40.0 ± 32.65 b 0.3 ± 0.14 b P. oxalicum (Po9) 20.0 ± 00.00 b 0.1 ± 0.08 b 25.0 ± 20.00 b 0.1 ± 0.10 c Control 95.0 ± 20.00 a 0.9 ± 0.30 a 95.0 ± 20.00 a 0.9 ± 0.15 a (a) The percentages of disease incidence were calculated after 100 days after transplanting as follows: disease incidence = No. of infected plants/No. of total plants × 100 (Brix and Zinkernagel, 1992). Values are means of four replicates with five onion seedlings each. The individual plants were rated for disease severity with a scale of 0 = healthy plants, 1 = slightly severe (yellowing of the leaves, reduced root system), 2 = moderately severe (yellowing and die-back of leaves, root system badly decayed), 3 = severe (complete yellowing of the plant, die-back of the leaves, semiwatery soft rot of scales and roots) and 4 = highly severe (completely dead plants, extensively decayed bulbs and roots). Disease severity was calculated according to the formula described by Zewide et al. (2007) with some modification: Disease severity = ∑ (Disease scale × Number of plants in each scale)/ Total number of plants × Highest disease scale. Values are means of four replicates with five onion seedlings each. (b) Means ± standard deviations within a column followed by the same letter are not significantly different by Duncan multiple range test at P < 0.05. Arcsine square root transformed data for disease incidence (%) and the nonparametric rank test for disease severity were conducted for statistic analysis; however, untransformed data are presented.

(Po9) was 11.3, 17.0, 8.5 and 16.0, 19.0, 12.5 mm, com- Effect of cultural filtrates of mycoparasites on scle- pared to the growth in control treatment being 90.0 and rotia germination. As shown in Table 4, the three my- 90.0 mm, respectively. Fourteen days after inoculation, coparasite cultural filtrates reduced the percentages of the inhibited colony of S. cepivora was usually compact germinated sclerotia. Filtrate of P. oxalicum (Po9) was the and dense and the hyphae were generally more branched most effective in reducing the percentage of germinated and no sclerotia were formed (Table 2). Obtained data sclerotia by 70.1%. It was followed significantly by Ch. are in accordance with earlier reports (Harrison and globosum (Chg6) and C. rosea (Cr12), where the percent- Stewart, 1988). Ch. globosum, C. rosea (syn. Gliocladium ages of reduction in sclerotia germination were 56.0 and roseum Bainier) and P. oxalicum have been shown to be 50.0%, respectively. A role in the production of cell wall biocontrol agents against several soil-borne plant patho- degrading enzymes such as β-1,3 glucanases, chitinases, gens (Soytong et al., 1999; Larena et al., 2003; Aggarwal proteases, cellulases, xylanases, esterases, alkaline phos- et al., 2004). Tathan et al. (2012) reported that Ch. globo- phatase and lipase by mycoparasites during interactions sum inhibited the spore production of Bipolaris oryzae with other fungi has been suggested (Tweddell et al., 1994). (Breda de Haan) Shoemaker by 87.94%. Characterization C. rosea (syn. Gliocladium roseum Bainier) produces tox- of antifungal metabolites of Ch. globosum was studied by ins and wall degrading enzymes affecting the germination Biswas et al. (2012), who reported that two out of five me- of sclerotia (Pachenari and Dix, 1980). An extracellular tabolites produced by Ch. globosum, viz. chaetoglobosin β-1,3-glucanase produced by Ch. globosum (Chg 2) was pu- and chaetomin, proved to be effective in suppressing the rified and characterized by Ahammed et al. (2012). growth of Bipolaris sorokiniana (Sacc.) Shoemaker, Fu- sarium graminearum Schwabe, Globisporangium ultimum Mycoparasites efficiency on onion white rot disease. (Trow) Uzuhashi, Macrophomina phaseolina (Tassi) Goid. Greenhouse experiments. The three mycoparasites exhib- and Thanatephorus cucumeris (A.B. Frank) Donk under ited the same trend during both seasons (Table 5). Appli- in vitro conditions. cation of the inoculum of sclerotial mycoparasites to the Journal of Plant Pathology (2017), 99 (2), 391-401 Elshahawy et al. 397

Table 6. White rot incidence (%) and severity in soil naturally infested with S. cepivora amended with sclerotial mycoparasites un- der field conditions.

(a) White rot incidence (%) and severity Mycoparasite Experiment 1 Experiment 2 Incidence (%) Severity Incidence (%) Severity Ch. globosum (Chg6) (b) 13.6 ± 1.10 c 0.1 ± 0.01 b 14.1 ± 1.10 c 0.1 ± 0.01 b C. rosea (Cr12) 17.5 ± 1.10 b 0.1 ± 0.01 b 17.0 ± 1.10 b 0.1 ± 0.01 b P. oxalicum (Po9) 12.2 ± 1.79 c 0.1 ± 0.01 b 13.3 ± 1.79 c 0.1 ± 0.01 b Control 67.5 ± 9.23 a 0.7 ± 0.08 a 65.2 ± 3.11 a 0.6 ± 0.08 a (a) The percentages of disease incidence were calculated at harvest (150 days after transplanting) as follows: Disease incidence = No. of infected plants/ No. of total plants × 100 (Brix and Zinkernagel, 1992). Harvested plants were subjected to examination of the root systems. White rot severity was evalu- ated on a scale of 0 to 6, where 0 = 0%, 1 = (1 to 14%), 2 =(15 to 35%), 3 = (36 to 64%), 4 = (65 to 85%), 5 = (86 to 99%), and 6 = 100% of the root system decayed. Disease severity was calculated according to the formula described by Zewide et al. (2007) with some modification: Disease severity = ∑ (Disease scale × Number of plants in each scale)/Total number of plants × Highest disease scale. Values are means of four replicates with 90 onion seedlings each. (b) Means ± standard deviations within a column followed by the same letter are not significantly different by Duncan multiple range test at P < 0.05. Arcsine square root transformed data for disease incidence (%) and the nonparametric rank test for disease severity were conducted for statistic analysis; however, untransformed data are presented. soil (artificially infested with S. cepivora Sc2) significantly and Trichoderma viride Pers. P. oxalicum reduced vascular reduced the incidence and severity of white rot disease wilts caused by Verticillium dahliae Kleb. and Fusarium compared to the control. No significant differences were oxysporum f. sp. lycopersici (Sacc.) Snyder & H.N. Hansen found between the mycoparasites. In the growing season under glasshouse and field conditions (Larena et al., 2003). of 2014/2015, P. oxalicum (Po9) recorded the lowest per- Hoitink and Boehm (1999) have reported several types of centage of disease incidence (20.0%), compared to the mechanisms that are used by biocontrol agents. These control (95.0%). Disease severity was also reduced to 0.1 include competition for nutrients and ecological niches, by P. oxalicum (Po9). Ch. globosum (Chg6) and G. roseum parasitism and production of cell-wall hydrolytic enzymes (Gr12) decreased the incidence and severity of white rot and/or of antifungal compounds. disease to 30.0%, 0.2 and 35.0%, 0.3, respectively. The obtained reduction in onion plants invaded by with S. ce- Defense enzyme activities. Inoculation of soil with pivora (Sc2) may be attributed to the high accumulative sclerotial mycoparasites induced increased production of inoculum potential of the mycoparasites introduced into defense enzymes in onion plants compared with the con- the root region, before sowing and throughout the grow- trol (Table 7). Ch. globosum (Chg6) treatment increased ac- ing season as well, where they have a direct impact on tivation of peroxidase enzyme by 163.6% over the control. sclerotia population. Similar explanation was reported by It was followed by P. oxalicum (Po9) and C. rosea (Cr12). Kay and Stewart (1994). Application of C. rosea (Cr12) increased activation of both polyphenoloxidase and chitinase by 174.5 and 163.9% Field experiments. Results of the two experiment sites over the control, respectively (Table 7). It was followed followed the same trend (Table 6). In experiment 1, data by Ch. globosum (Chg6) and P. oxalicum (Po9), where the indicate that soil treatment with the inoculum of scle- polyphenoloxidase and chitinase activities reached 115.7 rotial mycoparasites significantly reduced the incidence and 121.9%, respectively, over the control. The reduction and severity of onion white rot disease. P. oxalicum (Po9) in onion white rot disease incidence and severity may be and Ch. globosum (Chg6) gave the lowest percentages of due to an increase in the defense-related enzymes such as disease incidence (12.2 and 13.6%), in comparison with peroxidase, polyphenoloxidase and chitinase. The oxida- 67.5% for the control. C. rosea (Cr12) showed moderate tive enzymes play an important role in induced resistance efficiency, reducing the incidence of white rot disease to by oxidizing phenols to oxidized toxic products (quinine) 17.5%. No significant differences were found between my- which limit fungal activity. Peroxidases catalyze a num- coparasites in reducing disease severity of onion white rot. ber of reactions that fortify plant cell walls, including the Ch. globosum, P. oxalicum and C. rosea (syn. Gliocladium incorporation of phenolics into cell walls and lignifica- roseum Bainier) have been reported as effective in reduc- tions and suberization of plant cell walls. On the other ing seed rot and damping off caused by several seed- and hand, the chitinase enzymes play a role in plant defense soil-borne plant pathogens (Larena et al., 2003; Aggarwal against fungi by hydrolyzing their cell wall. The amount et al., 2004; Hung et al., 2015). In New Zealand, under significantly increases and plays a role in defense reaction controlled conditions, Ch. globosum provided an average of against fungal pathogen by degrading cell wall, because about 73% suppression of onion white rot over two years chitin is a major structural component of the cell walls (Kay and Stewart, 1994). They confirmed that the use of of many pathogenic fungi (Hammerschmidt et al., 1984; Ch. globosum is as effective as Trichoderma harzianum Rifai Kavroulakis et al., 2005; Chen et al., 2010; Jian et al., 2011). 398 Sclerotial mycoparasites as biocontrol agents Journal of Plant Pathology (2017), 99 (2), 391-401

Table 7. Effect of sclerotial mycoparasites on peroxidase, polyphenoloxidase and chitinase activities in onion plants grown in soil naturally infested with S. cepivora under field conditions.

(a) Enzyme activities Mycoparasite Peroxidase activity Polyphenoloxidase activity Chitinase activity Ch. globosum (Chg6) (b)0.290 ± 0.0048 a 0.440 ± 0.0048 b 1.556 ± 0.0032 b C. rosea (Cr12) 0.227 ± 0.0019 c 0.535 ± 0.0016 a 1.850 ± 0.0432 a P. oxalicum (Po9) 0.280 ± 0.0032 b 0.440 ± 0.0016 b 1.556 ± 0.0043 b Control 0.110 ± 0.0000 d 0.204 ± 0.0074 c 0.701 ± 0.0016 c (a) Peroxidase activity was expressed as the increase in absorbance at 470 nm/g fresh weight/minute. Polyphenoloxidase activity was expressed as the increase in absorbance at 475 nm/g fresh weight/minute. Chitinase activity was expressed as mM N-acetylglucosamine equivalent released/gram fresh weight/60 minutes at 540 nm. Values are means of four replicates. (b) Means ± standard deviations within a column followed by the same letter are not significantly different by Duncan multiple range test at P < 0.05.

Table 8. Effect of sclerotial mycoparasites on average plant height (cm), average number of leaves/plant and average plant biomass (g) of onion plants grown in soil artificially infested with S. cepivora in pots.

(a) Average growth parameters of onion plants grown in pots Mycoparasite 2014/2015 growing season Plant height (cm) No. of leaves/plant Plant biomass(g) Ch. globosum (Chg6) (b) 47.8 ± 0.38 b 6.3 ± 0.22 b 28.0 ± 0.73 c C. rosea (Cr12) 56.6 ± 1.38 a 6.7 ± 0.00 a 33.3 ± 0.99 a P. oxalicum (Po9) 56.3 ± 0.34 a 6.4 ± 0.28 b 30.5 ± 1.15 b Control 43.4 ± 0.16 c 4.8 ± 0.20 c 22.2 ± 0.32 d 2015/2016 growing season Mycoparasite Plant height (cm) No. of leaves/plant Plant biomass (g) Ch. globosum (Chg6) 47.4 ± 1.22 b 6.5 ± 0.20 a 29.2 ± 1.05 c C. rosea (Cr12) 56.7 ± 0.51 a 6.6 ± 0.00 a 32.9 ± 0.51 a P. oxalicum (Po9) 56.1 ± 0.30 a 6.3 ± 0.76 a 31.7 ± 2.07 b Control 42.3 ± 0.50 c 4.2 ± 0.00 b 20.6 ± 0.88 d (a) At the end of the experiment (100 days after transplanting), plant height (cm), number of leaves/plant and biomass (g) of alive plants was measured. Data collected from greenhouse experiments were analyzed separately for each growing season. Values are means of four replicates. (b) Means ± standard deviations within a column followed by the same letter are not significantly different by Duncan multiple range test at P < 0.05.

These results are also in agreement with those obtained growing season of 2014/2015, the treatment of infestation by De Cal et al. (1997). They reported that induction of with the highly pathogenic S. cepivora isolate (Sc2) with resistance in tomato plants was demonstrated as the main the inoculum of sclerotial mycoparasites significantly pro- mode of action of P. oxalicum against Fusarium oxysporum moted the growth of onion plants (Table 8). C. rosea (Cr12) f. sp. lycopersici; this resistance was also observed both had the highest effect, followed by P. oxalicum (Po9) and in sensitive and resistant cultivars, indicating the role of Ch. globosum (Chg6). The average plant height, number general resistance mechanism. Recently, Aggarwal (2015) of leaves and plant biomass after these treatments were investigated the important parameters of induced resis- 56.6, 56.3 and 47.8 cm; 6.7, 6.4 and 6.3; and 33.3, 30.5 and tance in wheat (Triticum aestivum) against B. sorokiniana 28.0 g, compared with 43.4 cm, 4.8 and 22.2 g for control, and Puccinia recondita Roberge ex Desm. using biocontrol respectively. Mycoparasites are thought to promote the agent Ch. globosum. Enhanced activities of defense-related growth of an onion plant grown under greenhouse condi- enzymes polyphenoloxidase, peroxidase, phenylalanine tions by at least two different mechanisms: (i) by reducing lyase and catalase revealed their role in induction of sys- Stromatinia injuries and (ii) by influencing plant physiol- temic resistance. The results indicate that the biocontrol ogy through mineral solubilization or hormone secretion, agents induced effective defense responses in wheat plants as reported by Moody and Gindrat (1977) and Aggarwal against B. sorokiniana and P. triticina. The reduced dis- (2015). ease incidence in wheat induced by Ch. globusum may be a result of cell wall strengthening through deposition of Field experiments. The effect of amending the soil with lignin and induction of defense enzymes. the inoculum of sclerotial mycoparasites on onion bulb yield was analyzed. Experiments were carried out at two Mycoparasites efficiency on onion growth and yield. sites, with similar outcomes (Table 9). In the first one, all Greenhouse experiments. Data in Table 8 showed that the tested mycoparasites significantly increased onion the three mycoparasites exhibited the same trend. In the bulb yield compared to control. Ch. globosum (Chg6) and Journal of Plant Pathology (2017), 99 (2), 391-401 Elshahawy et al. 399

Table 9. Effect of sclerotial mycoparasites on the yield of onion bulbs grown in soil naturally infested with S. cepivora under field conditions.

(a)Average weight (k/m2) of onion bulbs produced from the field Mycoparasite Experiment 1 Experiment 2 Average yield (kg/m2) Efficiency (%) Average yield (kg/m2) Efficiency (%) Ch. globosum (Chg6) (b) 5.6 ± 0.28 a 86.7 7.0 ± 0.56 a 75.0 C. rosea (Cr12) 4.2 ± 0.32 b 40.0 5.1 ± 0.16 c 27.5 P. oxalicum (Po9) 5.4 ± 0.28 a 80.0 6.4 ± 0.56 b 60.0 Control 3.0 ± 0.56 c 0.0 4.0 ± 0.32 d 0.0 (a) At the end of the experiment (150 days after transplanting), onion plants (bulbs with the tops of the plants) within each plot were weighed. Data collected from field experiments were analyzed separately for each experimental site. Values are means of four replicates. (b) Means ± standard deviations within a column followed by the same letter are not significantly different by Duncan multiple range test at P < 0.05.

P. oxalicum (Po9) recorded the highest efficiency, increas- ACKNOWLEDGEMENTS ing onion bulb yield by 86.7 and 80.0% over control, re- spectively (Table 9). C. rosea (Cr12) occupied the second This study was financially supported by The Affairs of rank, increasing onion bulb yield by 40.0% over control. Research Projects, National Research Centre. The ability of P. oxalicum to improve the seed germination and seedling growth of cabbage was reported by Teshima and Sakamoto (2006). Fungal biocontrol agents enhanced REFERENCES the plant growth parameters. C. rosea, P. oxalicum and Abd El-Razik A.A., Shatla M.N., Rushdi M., 1973. Studies on Ch. globosum influence plant growth through numerous the infection of onion plants by Sclerotium cepivorum Berk. mechanisms, which mainly include enhancing the solubi- Phytopathologische Zeitschrift 76: 108-116. lization of soil nutrients (Moody and Gindrat, 1977; Ag- Aggarwal R., 2015. Chaetomium globosum: a potential biocon- garwal, 2015), increasing root length and number of root trol agent and its mechanism of action. Indian Phytopathol- hairs to explore larger spaces of soil to absorb nutrients ogy 68: 8-24. (De Cal et al., 2000) and improving the production of Aggarwal R., Tiwari A.K., Srivastava K.D., Singh D.V., 2004. plant stimulatory compounds, such as growth hormones, Role of antibiosis in the biological control of spot blotch e.g. indole acetic acid, cytokinins, gibberellins, and zeatin (Cochliobolus sativus) of wheat by Chaetomium globosum. (Hyakumachi, 1994). Mycopathologia 157: 369-377. Ahammed S.K., Aggarwal R., Singh D.V., 2005. Morphological variability in different isolates of Chaetomium globosum. In- dian Phytopathology 58: 71-74. CONCLUSIONS Ahammed S.K., Aggarwal R., Sharma S., Gupta S., Bashyal B.M., 2012. Production, partial purification and character- In the present study, three fungal isolates from a total of ization of extra-cellular β-1, 3-glucanase from Chaetomium 30 were selected on the basis of their antagonistic activity globosum and its antifungal activity against Bipolaris soroki- against the most pathogenic isolate of S. cepivora (Sc2), and niana causing spot blotch of wheat. Journal of Mycology and identified as Chaetomium globosum (Chg6), Clonostachys Plant Pathology 42: 146-152. rosea (Cr12) and Penicillium oxalicum (Po9). These isolates Anonymous, 2015. Bulletin of the Agricultural Statistics. Min- colonized the surface of sclerotia of S. cepivora (Sc2), de- istry of Agriculture and Land Reclamation, Egypt, pp. 159. graded their outer rind and prevented their germination. Banks E., Edgington L.V., 1989. Integrated control of Sclero- Under greenhouse and field conditions, soil treatment by tium cepivorum Berk in organic soils. Canadian Journal of inoculum of these sclerotial mycoparasites significantly Plant Pathology 11: 268-272. reduced the incidence and severity of onion white rot dis- Barnett H.L., Hunter B.B., 1972. Illustrated Genera of Imper- ease. P. oxalicum (Po9) was the most effective, followed fect Fungi. Burgess Publ. Com., Minneapolis, pp. 241. by Ch. globosum (Chg6) and C. rosea (Cr12). Also, these Bashan Y., Okon Y., Henis Y., 1985. Peroxidase, polyphenol treatments increased vegetative growth parameters of the oxidase and phenols in relation to resistance against 214 survived onion plants in greenhouse and increased onion Pseudomonas syringae pv. tomato in tomato plants. Canadian Journal of Botany : 366-372. bulb yield in the field. Sclerotial mycoparasites caused an 65 Bell D.K., Wells H.D., Markham C.R., 1982. In vitro antago- enhancement of peroxidase, polyphenoloxidase and chitin- nism of Trichoderma species against six fungal plant patho- ase enzymes in onion plants grown in field. The levels of gens. Phytopathology 72: 382-379. protection provided by the tested sclerotial mycoparasites Biswas S.K., Aggarwal R., Srivastava K.D., Gupta S., Dureja P., represent practical potential for onion white rot control 2012. Characterization of antifungal metabolites of Chaeto- in fields. mium globosum Kunze and their antagonism against fungal plant pathogens. Journal of Biological Control 26: 70-74. 400 Sclerotial mycoparasites as biocontrol agents Journal of Plant Pathology (2017), 99 (2), 391-401

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Received September 5, 2016 Accepted February 7, 2017