The main goal of this study was enhancing the biocontrol activity of Trichoderma spp. (T. harzianum, T. koningii, T. viride, and T. virens) against Cephalosporium maydis, the cause of late wilt disease in maize. Five isolates of C. maydis were isolated from diseased maize plants, showing late wilt symptoms, and were collected from infected maize fields in Gharbia Governorate, Egypt. Pathogenicity test revealed that all C. maydis isolates were able to attack maize plants (cv. Baladi), which cause late wilt disease. Isolate 3 (Cm3) was the most virulent of them. In in vitro experiments, vegetative growth of the mycelium of C. maydis was highly inhibited after opposite sides’ treatment by Trichoderma species on Potato Dextrose Agar plates amended with Chlorella vulgaris extracts (cool and hot extracts) than unamended one. Formulation of C. vulgaris extracts and Trichoderma spp. were prepared. The formulations maintained the capacity of Trichoderma spp. to inhibit growth of the pathogen for up to 1 year when stored at both room temperature or at 7 °C. These formulations (3-day-old) were examined for biological control activities against late wilt disease of maize. Under greenhouse and field conditions, all treatments reduced late wilt incidence compared to the untreated control. Treatments involved Trichoderma spp., and C. vulgaris extracts were more effective than that used individually. Both of the C. vulgaris extracts, with each of T. virens and T. koningii, were the most effective treatments in this respect. Under greenhouse conditions, formulation treatments (C. vulgaris extracts and Trichoderma spp.) significantly increase the plant growth of maize plants, i.e., plant height and plant dry weight as compared to the non-treated control either in infested or in un-infested soil with C. maydis. Under field conditions, these formulations increased the grain yield as well as ear parameters as compared with either C. vulgaris extracts or Trichoderma spp. alone as well as non-treated control. This study suggests that the efficacy of Trichoderma spp. was enhanced with C. vulgaris extracts and these formulations can be developed as bio-fungicides for minimizing the late wilt disease caused by C. maydis in maize. Keywords: Chlorella vulgaris extract, Late wilt disease, Maize plants, Trichoderma spp., Biological control Background during tasseling as a rapid wilting of the lower leaves Maize, Zea mays L., is one of the most important cereal and develops to hollow and shrunken stalks with a dark crops worldwide. In Egypt, the cultivated maize area yellow-to-brown or black-stained pith (El-Shafey and reached about 88,000 ha that yielded almost 7.2 million Claflin 1999). The pathogen is a soil-borne vascular wilt metric tons of grains (Anonymous 2017). Black bundle disease that enters tissue of the root and colonizes the disease or late wilt, caused by Cephalosporium maydis, xylem (Sabet et al. 1970). Less commonly, this pathogen is one of the main economical and distributed maize dis- can be seed-borne (E1-Shafey et al. 1976) and may ir- eases in Egypt (Samra et al. 1963). This disease appears regularly cause decay of seed or pre-emergence damping-off under heavy inoculum pressure (Sabet et al. * Correspondence: email@example.com 1970). This fungus duplicates asexually and has not been Plant Pathology Department, National Research Centre, Giza, Egypt in perfect stage (Saleh and Leslie 2004). Large economic Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Elshahawy and El-Sayed Egyptian Journal of Biological Pest Control (2018) 28:48 Page 2 of 11 losses have been reported in Egypt by late wilt disease. Unit, National Research Centre, Giza, Egypt. The culti- In susceptible varieties, the disease affected 70% of the vation was performed, using a 1200-l open-plate photo- plants decreasing the grain yield by 40% (Labib et al. bioreactor. Microalgae nutrition was performed as 1975). Breeding of resistant varieties of maize is the most described by El-Sayed et al. (2015). Grown culture was effective method for controlling this disease (El-Shafey concentrated and dewatered by gravity. Purification of et al. 1988). Various bacteria and actinomycetes have the obtained biomass was performed by a series of pre- been evaluated as biocontrol agents against late wilt dis- cipitation by cooling centrifuge and washing it using tap ease (El-Mehalowy et al. 2004 and Ashour et al. 2013). water. This procedure was repeated several times to re- Little information has been cited in the literature on the move any excess of nutrients and mineral elements. The efficiency of Trichoderma spp. against late wilt disease. obtained biomass was dried at 45 °C within a circulated Trichoderma spp. isolated from Egyptian soil were used oven and then ground to a fine powder (Hassan et al. as a biocontrol for Colletotrichum dracaenophilum and 2015). Fusarium proliferatum, based on results of laboratory Hot (at 70 °C) and cool water extracts were produced trials (Morsy and Elshahawy 2016 and Elshahawy et al. by soaking 10% of microalgae biomass with distilled 2017a). It reduced disease caused by the soil-borne fun- water and solicited using ultrasonic homogenizer. After gus Stromatinia cepivora (Berk.) and induced plant re- homogenization, the extracted materials were obtained sistance in onion plants when applied to soil (Elshahawy by filtration through filter paper (Whatman no. 1). The et al. 2017b). extracts were freeze-dried and sieved in a refrigerator The microalgae, Chlorella vulgaris known as fresh- until used. Total sugars were determined according to water algae, is one of the most remarkable green micro- Dubois et al. (1956). Polysaccharides were determined in algae. There are several applications and potential extracts. Firstly, freeze-dried extracts were sequentially benefits of this microalga such as biofuels, human nutri- treated by petroleum ether and chloroform to remove tion, animal feed, wastewater treatment, and agrochem- oiled materials. Absolute ethanol was used to precipitate ical applications (Safi et al. 2014). C. vulgaris contains polysaccharides. Forty milliliters of absolute ethanol was high amounts of micro- and macronutrients, proteins, added gradually to 10 ml of water extracts (1:20 w/v). and carbohydrates (Wake et al. 1992). It is used as The mixtures were left overnight into the refrigerator bio-fertilizer and soil conditioner in agriculture systems and then centrifuged (5500 rpm for 10 min). The precip- (Song et al. 2005). Algal extract can be partially substi- itated polysaccharides were dried using a freeze drier tuting micronutrient foliar fertilizers and best to be and determined by gas-liquid chromatography (GLC). complementary portion of the spray solution (Shabaan 2010). Soil fertility can be improved by entrapping some Trichoderma species rhizosphere bacteria with Chlorella (Raposo and Morais Four Trichoderma species, viz., T. harzianum, T. konin- 2011). Newly, the consortium of C. vulgaris, Azotobacter gii, T. viride, and T. virens, were obtained from Plant sp., and Anabaena variabilis was found to increase ger- Pathology Department, NRC, Egypt. The Trichoderma mination and plant growth of rice, and it is suggested as species were isolated from Egyptian soil, identified, and a bio-fertilizer and a bio-stimulator for crops as reported evaluated for their efficiency in previous study (Elsha- by Zayadan et al. (2014). hawy et al. 2016). The present study was conducted to evaluate the effi- ciency of Trichoderma species either alone or mixed C. maydis isolates with the C. vulgaris extracts on the incidence of maize Maize plant samples, showing typical late wilt symp- with late wilt under greenhouse and field conditions. toms, were collected from naturally infected fields lo- cated at Gharbia Governorate, Egypt. Isolation of C. Materials and methods maydis was carried out according to Samra et al. (1963). Experimental site Stems of diseased maize plants were cut into small This study was carried out at the Agriculture and Bio- pieces, and the surface was disinfected with 0.5% sodium logical Division, National Research Centre (NRC), as hypochlorite for 3 min and then washed thoroughly with well as within a disease nursery field located at Gharbia sterilized water. The disinfected stem pieces were dried Governorate, Egypt, during the 2016 growing season. between folds of sterile filter papers, then plated onto potato dextrose agar (PDA) medium supplemented with Freshwater microalgae, Chlorella vulgaris, and preparation 0.2% yeast extract and incubated at 28 ± 2 °C for 72 h. of extracts Hyphal tip isolation technique was employed to obtain C. vulgaris was isolated from freshwater Nile River at the fungus isolation in pure cultures. C. maydis was Cairo Governorate, Egypt (El-Sayed et al. 2001). This identified according to morphological and cultural fea- strain was massively produced at Algal Biotechnology tures using the descriptions of Samra et al. (1963) and Elshahawy and El-Sayed Egyptian Journal of Biological Pest Control (2018) 28:48 Page 3 of 11 Ainsworth and James (1971). Five isolates of C. maydis Trichoderma spp. alone, the inhibitor effect of T. harzia- were obtained from diseased maize plants and kept at num, T. koningii, T. viride,and T. virens against the 4 °C for further studies. growth of the most virulent isolate of C. maydis (isolate Cm3) was studied, using the method described by Bell et Inoculum preparation and determination of pathogenicity al. (1982). Petri plate containing PDA medium supple- The isolates of C. maydis were grown into 250 ml potato mented with 0.2% yeast extract was inoculated on one side dextrose broth medium supplemented with 0.2% yeast with a 5-mm mycelial disc from a 7-day-old culture of the extract in 500 ml Erlenmeyer flasks. After sterilization, tested Trichoderma spp. The opposite side was inoculated flasks were inoculated with each of the different isolates with a disc of C. maydis, and the plates were incubated at of C. maydis and then incubated at 28 ± 2 °C for 2 weeks. 28 ± 2 °C. Plates inoculated with a disc of C. maydis alone The flasks were thoroughly shaken, and about 20 ml of were used as control. Four replicate plates were made for the suspension was poured into 1-l glass bottles contain- each test fungus as well as for the control. Colony radius ing wet autoclaved crushed grain sorghum up to two of C. maydis was recorded when the control plates thirds of its capacity. The inoculated glass bottles were reached full growth. On the other hand, the effect of C. then kept at 28 ± 2 °C for 4 weeks. Pathogenicity test of vulgaris water extracts on the antagonistic activity of Tri- the obtained isolates of C. maydis was conducted on a choderma spp. against C. maydis was carried out, using susceptible maize cultivar Baladi. Disinfested grain seeds PDA plates amended with each of cool or hot extracts. were planted in pots (30 cm in diameter) containing Ten milliliters of each extract was filtered through a sterile autoclaved clay loam soil (6 kg/pot), infested with the in- 0.22-μm Millipore filter directly into 190 ml molten PDA. oculum of different isolates. Seed disinfestations were The medium was poured into sterile Petri plates and carried out by soaking seeds in 5% sodium hypochlorite cooled at room temperature. The amended plates were solution for 3 min and rinsed in sterile water. Pots and used for dual culture test described before. Plates soil were treated 2 weeks before planting by autoclaving amended with cool extract, hot extract, and sterile distilled the soil and soaking the pots in 7% formalin solution for water and inoculated with a disc of C. maydis by itself 3–5 min. Soil infestation was carried out 7 days before were used as control. Four replicate plates were used for planting by mixing 180 g of inoculum to the soil in every each treatment as well as for controls. Colony radius of C. pot and mixed thoroughly to ensure equal distribution maydis was recorded when the control plates reached full of fungal propagates, followed by irrigation. Each pot growth. The reduction in the growth of C. maydis was cal- was seeded with eight grain seeds of the Baladi cv., and culated, using the following formula: plants were thinned to three plants per pot. Six pots were used for each isolate, and a non-inoculated treat- Growth reductionðÞ % ¼½ ðÞ C−T =C 100: ment was used as control. Nitrogen fertilizer in the form of urea (46% N) was added at 500 mg N/kg soil, 30 days after planting, and plants were irrigated when necessary. where C is the average linear growth of C. maydis in Percentage of dead plants due to late wilt infection was control and T is the average linear growth of C. maydis calculated 80 days after planting. Disease symptoms in biocontrol agent treatment. began to appear approximately 60 days after sowing. Pots were examined at weekly intervals thereafter and Development of Trichoderma–C. vulgaris extract symptomatic plants removed when they were identified. formulation Fungal isolates were recovered from internodes of symp- Trichoderma spp. propagules tomatic plants to demonstrate Koch’s postulates. Among Trichoderma harzianum, T. koningii, T. viride,and T. the tested isolates, the highest aggressive isolate was se- virens were grown on a PDA medium at 25 ± 2 °C for lected and used throughout the present study. The 10 days. Afterwards, the mycelium with the spores was maize plants were harvested at 80-day age by mulching scraped from Petri plates and mixed with sterilized dis- the plants from the pots. The length of plants and their tilled water (20 ml/plate) in a blender. The suspension dry weight were determined. The harvested plants were was adjusted by a hemocytometer slide to 10 propa- dried at 70 °C till constant weight, and the dry weight gates/ml. per plant was recorded. Laboratory experiments Preparation of C. vulgaris extracts Antagonistic activity tests Each of cool or hot extracts of C. vulgaris was prepared Testing the antagonistic activities of Trichoderma spp. individually. Two hundred and fifty milliliters of each which uses either alone or in combination with C. vulgaris extract was filtered through a sterile 0.22-μm Millipore extracts against C. maydis was carried out. In the case of filter directly into a 500-ml sterile conical flask. Elshahawy and El-Sayed Egyptian Journal of Biological Pest Control (2018) 28:48 Page 4 of 11 Incorporation of Trichoderma spp. to C. vulgaris extracts Agriculture and Biological Division, National Research Propagule suspension (10 propagates/ml) of each of Centre, Egypt. The experiment was carried out in a ran- Trichoderma spp. were individually incorporated into domized complete block design with four replicates. The sterilized C. vulgaris extracts under aseptic conditions at most virulent isolate of C. maydis (isolate Cm3) was the rate of 10 ml of suspension per 90 ml extract and used. Seed disinfections were carried out by soaking thoroughly shacked on a rotatory shaker at 70 rpm for seeds in 5% sodium hypochlorite solution for 3 min, 6 h. Each Trichoderma–C. vulgaris extract was first rinsed in sterile water. Pots (30 cm in diameter) and soil stored at room temperature for 3 days to increase the were treated 2 weeks before planting by autoclaving the initial population of Trichoderma spp., and then, they soil and soaking the pots in 7% formalin solution for 3– were applied. 5 min. Soil infestation was carried out 7 days before planting by mixing 180 g of C. maydis inoculum to the Population dynamics of Trichoderma spp. on C. vulgaris soil in every pot (6 kg soil/pot), followed by irrigation. extracts Disinfected maize grains (Baladi cv.) were soaked in each The viability of Trichoderma spp. in C. vulgaris extracts treatment at the rate of 100 ml/100 grain in 250-ml Er- was determined at 3, 60, 120, 180, 240, 300, and 360 days lenmeyer flasks. Control of grains was soaked in sterile after storage (DAS) of room temperature (27 ± 2 °C). For distilled water only. Few drops of Tween-80 were added the study of the potentiality of 7 °C storage conditions to improve adhesive. Flasks were incubated at 25 °C on a on the viability of the Trichoderma spp. in C. vulgaris rotary shaker at 70 rpm for 6 h to allow treatment mate- extracts, they were first stored at room temperature for rials to adhere to seeds. After incubation, excess inocu- 3 days to increase the initial population of Trichoderma lum was removed and grains were left to air-dry for spp. Initial determination of population of Trichoderma 30 min at room temperature and then immediately spp. was made at 3 DAS at room temperature, and later planted in the infected and/or un-infected potted soil samples were made at 60, 120, 180, 240, 300, and 360 (Ashour et al. 2013). Each pot was seeded with eight DAS at 7 °C. Serial dilutions of formulation samples grain seeds, and the plants were thinned to three plants. were used to determine the number of Trichoderma spp. The abovementioned treatments were applied to soil in propagules found on C. vulgaris extracts by the plate the pots with irrigation water at three equal doses count technique using selective media (Johnson et al. (30 ml per pot) each 10 days. Six pots were used for 1960). Thus, the blended 1 ml of formulation was trans- each treatment as well as control. Nitrogen fertilizer in ferred to bottles containing 99 ml of sterilized distilled the form of urea (46% N) was added at the rate of water under aseptic conditions. The bottles were shaken 500 mg N/kg soil, 30 days after planting, and the plants using a mechanical shaker for 15 min. Serial dilutions of were irrigated when necessary. fresh suspension were prepared for each Trichoderma The following treatments were used in soil infected spp. in C. vulgaris extract sample under sterile condi- and non-infected with late wilt pathogen: (1): T. harzia- tions. A portion of 1.0 ml formulation suspension from num (10 × 10 propagates/ml sterile distilled water), (2): −4 4 the dilution 10 was transferred to four sterile Petri T. koningii (10 × 10 propagates/ml sterile distilled plates. Rose Bengal streptomycin-selective medium was water), (3): T. viride (10 × 10 propagates/ml sterile dis- used for growing Trichoderma spp. colonies after 4 days tilled water), (4): T. virens (10 × 10 propagates/ml sterile of incubation at 25 ± 2 °C (Metcalf 1997). This medium distilled water), (5): T. harzianum (10 × 10 propagates/ consisted of 2.0 g of (NH ) SO , 4.0 g of KH PO , 6.0 g ml cool water extract of C. vulgaris), (6): T. koningii 4 2 4 2 4 of Na HPO , 0.2 g of Fe·SO 7H O, 1 mg of CaCl ,10 μg (10 × 10 propagates/ml cool water extract of C. vul- 2 4 4 2 2 of H BO ,10 μg of MnSO ,70 μgofZnSO , 1 l of dis- garis), (7): T. viride (10 × 10 propagates/ml cool water 3 3 4 4 tilled water, 20 g agar, and 5 g of cellulose powder extract of C. vulgaris), (8): T. virens (10 × 10 propa- (Sigma), adjusted to pH 4.0 before autoclaving. After the gates/ml cool water extract of C. vulgaris), (9): T. harzia- medium cooled to 70 °C, 0.05 g of streptomycin sulfate num (10 × 10 propagates/ml hot water extract of C. and 0.016 g of rose Bengal were added. vulgaris), (10): T. koningii (10 × 10 propagates/ml hot water extract of C. vulgaris), (11): T. viride (10 × 10 propa- Greenhouse experiments gates/ml hot water extract of C. vulgaris), (12): T. virens A pot experiment was conducted to evaluate the influ- (10 × 10 propagates/ml hot water extract of C. vulgaris), ence of Trichoderma spp. treatments alone or formu- (13): Cooled water extract of C. vulgaris, (14): Heat water lated on C. vulgaris extracts on the incidence of maize extract of C. vulgaris,(15): Control. late wilt as well as on growth parameters of maize plant Percentage of dead plants due to late wilt infection in soil infected and non-infected with late wilt pathogen. was recorded 80 days after planting. Vegetative growth The experiment was conducted in the summer season of parameters, i.e., plant height and dry weight, were also 2016 at the greenhouse of Plant Nutrition Department, recorded as previously described. Elshahawy and El-Sayed Egyptian Journal of Biological Pest Control (2018) 28:48 Page 5 of 11 Field experiments extract represented the maximum figure of total sugars The effect of Trichoderma spp. treatments alone or for- (16.42 and 12.31%) of total carbohydrates. GLC analysis mulated on C. vulgaris extracts on the incidence of of each fraction is listed in Table 1. The most abundant maize late wilt as well as on yield of maize plant was sugars are galactose, mannose, rhamnose, and glucose studied under field conditions in a disease nursery at that reached more than 10% of total carbohydrates. Con- Gemmiza Research Station, Plant Pathology Research In- cerning such chemical structure, growth conditions (out- stitute, Agriculture Research Center, Gharbia Governor- door mass production) markedly affected it to form a ate, Egypt, during the 2016 growing season. This nursery rigid cell wall. This is in agreement with Cheng et al. was infested artificially with the four clonal lineages of (2011), who described that the chemical composition of C. maydis found in Egypt that causes late wilt of maize the cell wall in Chlorella variabilis NC64A was impacted and commonly used in Egyptian maize breeding pro- by cultivation conditions such as uronic acid, neutral grams (Zeller et al. 2002). Maize grains cv. Baladi were sugar, and amino sugar in the cell wall when cultivated used in this study. The abovementioned treatments in in diverse sources and concentrations of nitrogen. Un- greenhouse were involved in field experiments. Disin- bending cell walls of Chlorella species contain mannose fected maize grains (Baladi cv.) were soaked in each as a major sugar component. Numerous polysaccharides treatment at the rate of 100 ml/100 grain. Control grains contained phosphate, carboxylic, and/or ester sulfuric were soaked in sterile distilled water only. Randomized groups in the molecular structure (Nelson and Cox complete block arrangement in three replicate plots was 2008). These polysaccharides, in a pure form, are pres- used. Each replicate included three ridges of 4.5-m ently the most commercial protectors for plants against length and 0.7-m width for each ridge, i.e., the experi- pathogens (Stadnik and Freitas 2014). mental plot area was 3.15 m . Thirteen maize plants for each treatment were used in each replicate. Grains were C. maydis isolation sown in holes (five holes/ridge with three grains/hole); Five isolates of C. maydis obtained from infected maize thereafter, they were thinned to one plant/hole. The plants were studied on a susceptible cultivar Baladi. For abovementioned treatments were also applied before ir- recording infection percentage by late wilt, typical dis- rigation with water at three equal doses (10 ml per hole) ease symptoms formerly described by Samra et al. each 15 days. Irrigation, recommended fertilizer levels, (1963) were observed on infected plants. Koch’s postu- and agronomical practices were used as usual. Disease lates were demonstrated for all C. maydis isolates recov- incidence of late wilt as infection percentage was re- ered from infected maize plants in the field. All C. corded 110 days after sowing. Quantitative maize yield maydis isolates were examined for pathogenicity toward and qualitative maize yield, i.e., ear length, ear diameter, maize plants in greenhouse. Results in Table 2 showed no. of rows per ear, no. of kernels per row, no. of kernels that the five isolates were capable of causing late wilt per ear, and 100-kernel weight, were evaluated during disease and were potentially pathogenic in greenhouse harvest period. assay system. Non-inoculated plants (control) did not develop late wilt symptoms. Data also showed that the Statistical analysis tested C. maydis isolates were statistically differed in Statistical analysis of data was conducted, using SPSS their aggressiveness toward maize plants where disease software version 14.0. Percent data of disease incidence percentages varied between 65.0 and 78.2%, at 110 days were statistically analyzed after arcsine square root after sowing. The most virulent isolate was Cm3, transformation; however, untransformed data are pre- sented. Analysis of variance was determined, and the Table 1 Intercellular saccharide content (% dw from total mean values were compared by Duncan’s multiple range carbohydrates) of Chlorella vulgaris cool and hot extract test at P < 0.05. Saccharide % content (dry weight from total carbohydrates) Cool Hot Results and discussion Galactose 25.5 26.30 Chemical analysis of Chlorella extract Mannose 16.3 11.90 Chlorella as the freshwater microalgae is considered the Rhamnose 14.5 18.20 most useful green algae (Liu et al. 2016). It contains li- Glucose 13.4 10.60 popolysaccharides which differ from Gram-negative bac- teria because chlorella has no endotoxins (Stewart et al. Arabinose 09.7 10.70 2006). Dried biomass total sugars of C. vulgaris repre- Xylose 06.6 08.90 sented 9.7% + 0.12. Out of this content, soluble sugar Fructose 02.9 02.96 content, which is determined by weight, varied between Ribose 02.8 02.03 the two fractions (cold and hot extracts). Mostly, hot Elshahawy and El-Sayed Egyptian Journal of Biological Pest Control (2018) 28:48 Page 6 of 11 followed by Cm4, while the isolates Cm1, Cm2, and caused the die back rapidly of fungal colony. Tricho- Cm5 were the least ones. These findings are in agree- derma spp. produce their biocontrol action against fun- ment with those obtained by Ali (2000). García-Carneros gal phytopathogens either indirectly by competing for et al. (2012) found that the initial incidence of late wilt nutrients and space or indirectly by mechanisms such as symptoms in maize plants depends on the isolate of C. antibiosis and mycoparasitism (Benítez et al. 2004). The maydis and on the maize variety and the final severity of additive effect of microalgae extracts to Trichoderma the aboveground symptoms only depends on the fungal species in dual culture may be due to its bio-stimulators isolate. The pathogenic differences among the tested iso- and immunity effects. These microalgae produce lates may be due to the genetic diversity among them. growth-promoting regulators, vitamins, amino acids, Zeller et al. (2002) found that the four phylogenetic line- polypeptides, and polymers such as exo-polysaccharides ages of C. maydis differed in their virulence and com- (Singh et al. 2005). Wake et al. (1992) reported that petitiveness toward maize plants grown under freshwater microalgae as C. vulgaris contain high greenhouse conditions. A highly negative correlation amounts of micro- and macronutrients (metabolites) as was observed among infection percentages incited by C. proteins and carbohydrates. These bio-fertilizers en- maydis isolates and each of plant height and dry weight hance Trichoderma growth and subsequently its antag- of maize plants after 110 days. These results are in har- onistic agent production. On the other hand, the mony with the findings of Alhanshoul (2015) who re- reduction of C. maydis growth caused by C. vulgaris ex- ported that infection with C. maydis isolates slightly tracts may be due to the low saprophytic behavior of C. reduced seed germination, plant height, and dry weight maydis (Sabet et al. 1970) that minimizes its growth on of plants. PDA amended with algae extracts. Antagonistic activity of Trichoderma spp. and C. vulgaris Population dynamics of Trichoderma spp. on C. vulgaris extracts against C. maydis extracts Four Trichoderma species were tested alone or in com- The results of the effect of cool and heat extracts of C. bination with C. vulgaris extracts for antagonistic activ- vulgaris-based liquid formulations on the population dy- ity against C. maydis, using the dual culture technique namics of Trichoderma spp. at room temperature indi- (Table 3). All Trichoderma species treatments inhibited cated that the algae extracts supported the highest the growth of C. maydis in dual culture compared to population of Trichoderma spp. during the DAS sampled control. Data indicated that T. harzianum, T. viride, T.virens, and T. koningii reduced the growth of C. may- Table 3 Antagonistic activity of Trichoderma spp. alone or in dis by 63.3, 50.0, 75.6, and 70.9%, respectively, when combination with Chlorella vulgaris extract against the linear used alone, while the reduction attained to 81.1 and growth of Cephalosporium maydis 80.0, 61.1 and 60.4, 87.6 and 88.0, and 85.8 and 86.9% Treatment Linear growth and growth reduction of C. maydis when they were used in combination with cool and hot Linear growth (cm) Reduction (%) extracts of C. vulgaris, respectively. Trichoderma species T. harzianum (Th) 3.30 ± 0.20d 63.3 showed rapid growth, outcompeting the pathogen for space and nutrients. After the Trichoderma species T. viride (Tv) 4.50 ± 0.15c 50.0 growth meets the C. maydis colony, it would inhibit fur- T. virens (Tvs) 2.20 ± 0.12f 75.6 ther growth of the hyphal tips of the pathogen and T. koningii (Tk) 2.62 ± 0.05e 70.9 Cool extract + Th 1.70 ± 0.12g 81.1 Cool extract + Tv 3.50 ± 0.15d 61.1 Table 2 Virulence of Cephalosporium maydis isolates on maize cv. Baladi under greenhouse conditions Cool extract + Tvs 1.12 ± 0.04h 87.6 Isolate Disease Plant’s vigor Cool extract + Tk 1.28 ± 0.03h 85.8 incidence Plant height (cm) Plant dry weight (g) Hot extract + Th 1.80 ± 0.12g 80.0 (%) C. maydis (Cm1) 67.2 ± 0.37c 66.2 ± 0.37bc 29.2 ± 0.48b Hot extract + Tv 3.56 ± 0.16d 60.4 C. maydis (Cm2) 65.0 ± 1.00d 64.6 ± 0.40c 27.0 ± 0.31c Hot extract + Tvs 1.08 ± 0.04h 88.0 C. maydis (Cm3) 78.2 ± 0.37a 59.4 ± 0.60d 23.6 ± 0.50d Hot extract + Tk 1.18 ± 0.04h 86.9 C. maydis (Cm4) 74.4 ± 1.16b 66.8 ± 0.37b 28.0 ± 0.31bc Cool extract 7.88 ± 0.08b 12.4 C. maydis (Cm5) 67.6 ± 0.60c 66.6 ± 0.24bc 26.8 ± 0.37c Hot extract 7.64 ± 0.17b 15.1 Control 00.0 83.4 ± 1.40a 34.8 ± 0.20a Control 9.00 ± 0.00a 00.0 The presented data are the mean ± standard errors, and the letters show The presented data are the mean ± standard errors, and the letters show significance at P ≤ 0.05 significance at P ≤ 0.05 Elshahawy and El-Sayed Egyptian Journal of Biological Pest Control (2018) 28:48 Page 7 of 11 (data not shown). The population of Trichoderma spp. Table 4 Effect of Trichoderma spp. alone or in combination with Chlorella vulgaris extract on the incidence of late wilt of found on cool and heat extracts of C. vulgaris followed a maize grown under greenhouse and field conditions fluctuating trend with the DAS sampled. The initial popu- Treatment Late wilt incidence (%) lation (3 days) of Trichoderma spp. was increased at 60 DAS. At 120 DAS, the population recovery in all the Tri- Greenhouse experiment Field experiment choderma spp. was significantly increased. Thereafter, dur- T. harzianum (Th) 45.2 ± 0.86c 31.8 ± 0.48c ing the period of 180 to 300 DAS, the population of T. viride (Tv) 37.6 ± 0.51d 28.6 ± 0.50d Trichoderma spp. was declined progressively, and at 360 T. virens (Tvs) 33.6 ± 0.24e 23.8 ± 0.37e DAS, a significant reduction of more than four- to nine- T. koningii (Tk) 30.2 ± 0.20f 24.6 ± 0.24e fold was recorded. The results of the population dynamics Cool extract + Th 30.0 ± 0.31f 20.8 ± 0.48f of Trichoderma spp. in extracts of C. vulgaris,when the li- Cool extract + Tv 27.6 ± 0.24g 16.4 ± 0.40g quid formulations were stored at 7 °C, showed that there was a slow and progressive decline of the antagonist popu- Cool extract + Tvs 23.2 ± 0.20h 12.8 ± 0.48h lations in the bioformulations from 120 DAS to 180, 240, Cool extract + Tk 23.8 ± 0.37h 12.4 ± 0.40h 300, and 360 DAS. Still, the population recovered was Hot extract + Th 27.6 ± 0.24g 17.6 ± 0.40g much greater as compared to that recorded during the Hot extract + Tv 30.0 ± 0.31f 19.6 ± 0.74f same period under room temperature storage conditions. Hot extract + Tvs 21.0 ± 0.31i 12.4 ± 0.40h These formulations maintained the capacity of Tricho- Hot extract + Tk 22.4 ± 0.24hi 12.4 ± 0.40h derma spp. to inhibit growth of the pathogen for up to 1 year when stored at both room temperature or at 7 °C Cool extract 57.4 ± 0.81b 46.8 ± 0.48a (data not shown). Hot extract 57.2 ± 0.37b 43.8 ± 0.20b Control 73.4 ± 1.66a 47.6 ± 0.24a Efficiency of treatments on maize late wilt disease The presented data are the mean ± standard errors, and the letters show After obtaining positive reaction of using microalgae significance at P ≤ 0.05 cool or hot water extracts with Trichoderma spp. in con- trolling of C. maydis, the experiments were applied under greenhouse and field conditions. This confirms extracts caused more reduction in maize late wilt com- the efficiency of applying C. vulgaris extracts as growth pared to the individual treatments of Trichoderma spp. promoters and biocontrol agents. Under greenhouse or C. vulgaris extracts. However, the combined treat- conditions, data presented in Table 4 showed that seed + ment of C. vulgaris extract with T. virens and/or T. soil treatment with Trichoderma spp. either alone or in koningii gave the highest reduction in late wilt incidence, combination with C. vulgaris extracts significantly re- being 73.1 and 74.0%, respectively, in comparison to duced the infection percentage with late wilt disease treatment with T. virens or T. koningii each alone, being compared to check treatment (73.4% infection) under 50.0 and 48.3% reduction, respectively. The lowest re- artificial soil infestation. Treatment with Trichoderma duction in disease incidence was recorded by C. vulgaris spp. formulated on C. vulgaris extracts gave the highest extract treatments only (being 1.7 and 8.0% reduction). effect in reducing infection percentages compared to The antagonistic effect of Trichoderma strains against Trichoderma spp. alone. Among the Trichoderma spp., soil-borne fungi was recently emphasized by Elshahawy T. virens formulated on hot extract of C. vulgaris, et al. (2017b). The reported data describe, for the first followed by T. virens formulated on cool extract and T. time, control of the pathogen, using Trichoderma strains koningii formulated on cool extract, were the effective in Egypt. The ability of Trichoderma strains to inhibit C. treatments in reducing infection percentages, being 72.1, maydis pathogen is noteworthy since the suppression 68.3, and 67.6% reduction in disease incidence, respect- obtained is a result of seed + soil treatment. Addition of ively. Other Trichoderma spp. treatments used either Trichoderma strains with C. vulgaris extracts increased alone or in combination with C. vulgaris extracts their effect against C. maydis. These results suggest that showed moderate effect. Treatment with C. vulgaris ex- C. vulgaris extracts stimulate the inhibitory activity of tracts alone showed the lowest effect in reducing the in- Trichoderma strains. This may be due to that C. vulgaris fection percentage with late wilt. extracts are considered as absorbed agents into plants In the field experiment, treatment of seed and soil for increasing of disease and stress resistance (Abd with Trichoderma spp. either alone or in combination El-Motty et al. 2010). Amendment of Trichoderma with C. vulgaris extracts showed significant reduction in strains with C. vulgaris extracts could increase the plant maize infection with late wilt compared to check plants protection by supporting the growth of Trichoderma (47.6% infection) as presented in Table 4. The combin- strains and stimulating the useful metabolite production ation treatments of Trichoderma spp. with C. vulgaris which may help antagonistic activity. Degani et al. Elshahawy and El-Sayed Egyptian Journal of Biological Pest Control (2018) 28:48 Page 8 of 11 (2015) reported that many plant growth promoters such treatments of T. virens and T. koningii with hot extract as hormones, auxin (indole-3-acetic acid), and cytokinin of C. vulgaris, followed the previous treatments in their (kinetin) were produced by higher levels, when using C. effect on plant height, being 102.2 and 102.8 cm for vulgaris extracts, suppressing C. maydis in culture media plants grown in infested soil as well as 115.8 and and in a detached root assay. 116.4 cm when grown in un-infested soil, respectively. Other treatments showed moderate effects. In concern Efficiency of treatments on maize growth in greenhouse to plant dry weight, results presented in Table 4 showed and on maize yield in field that all treatments recorded significant increment in dry Greenhouse experiment weight of maize plants grown either in infested or in Data presented in Table 5 showed that the seed + soil un-infested soil compared to check plants (24.2 and treatment with Trichoderma spp., either alone or in 33.8 g, respectively). Likewise, combined treatments be- combination with C. vulgaris extract, significantly pro- tween Trichoderma spp. and microalgae extracts signifi- moted plant growth compared to check treatment, cantly increased dry weight of plants in comparable with whether grown in infested or un-infested soil. Moreover, the individual treatments for plants grown either in Trichoderma spp. formulated with C. vulgaris extract infested or in un-infested soil. However, the combined caused significant increment in maize growth parame- treatments of T. virens and T. koningii with cool extract ters compared to treatment of Trichoderma spp. alone. of C. vulgaris gave the highest dry weight of plants Regarding plant height, data indicated that all treatments grown either in infested soil (49.4 and 50.6 g, respect- significantly increased plant height in either infested or ively) or in un-infested soil (58.4 and 58.4 g, respect- un-infested soil, being 60.4 and 78.41 cm, respectively. ively). The treatment of Trichoderma spp. and algae Combined treatments of Trichoderma spp. and microal- extracts had moderate effect on plant dry weight either gae extracts increased plant height when plants were grown in infested soil or in un-infested soil. The effect grown, either in infested or in un-infested soil compared of bio-fertilization, using microalgae extracts, was sug- to individual treatments. However, the combined treat- gested for increasing the growth parameters of many ment of T. virens and T. koningii with cool extract of C. plants. This is likely due to the biochemical composition vulgaris caused the highest plant heights when plants of microalgae extracts which are rich in essential nutri- were grown either in infested soil (103.6 and 106.4 cm) ents for plant growth as nitrate reductase, nitrogenase, or in un-infested soil (119.6 and 118.0 cm) in compari- and minerals. The impact of foliar feeding by water ex- son to the individual treatments. The combination tracts of C. vulgaris on growth, nutrient levels, and yield Table 5 Effect of Trichoderma spp. alone or in combination with Chlorella vulgaris extract on maize plant growth characters under infested and un-infested soil with Cephalosporium maydis Treatment Plant’s vigor Un-infested soil Infested soil Plant height (cm) Plant dry weight (g) Plant height (cm) Plant dry weight (g) T. harzianum (Th) 90.4 ± 0.24j 38.4 ± 0.24h 83.6 ± 0.24j 33.4 ± 0.24i T. viride (Tv) 88.2 ± 0.20k 36.4 ± 0.24i 84.0 ± 0.54j 31.4 ± 0.24j T. virens (Tvs) 91.6 ± 0.24i 40.4 ± 0.24g 88.4 ± 0.24h 33.8 ± 0.20hi T. koningii (Tk) 93.4 ± 0.24g 41.4 ± 0.24f 87.4 ± 0.24i 34.2 ± 0.20h Cool extract + Th 103.4 ± 0.24d 50.2 ± 0.20c 100.6 ± 0.40d 43.4 ± 0.24d Cool extract + Tv 100.0 ± 0.54f 50.4 ± 0.24c 100.8 ± 0.37d 44.4 ± 0.24c Cool extract + Tvs 119.6 ± 0.24a 58.4 ± 0.24a 103.6 ± 0.24b 49.4 ± 0.24b Cool extract + Tk 118.0 ± 0.31b 58.4 ± 0.24a 106.4 ± 0.24a 50.6 ± 0.24a Hot extract + Th 103.8 ± 0.20d 50.8 ± 0.37c 96.0 ± 0.31f 42.6 ± 0.24e Hot extract + Tv 101.4 ± 0.40e 50.4 ± 0.24c 97.2 ± 0.20e 43.0 ± 0.31de Hot extract + Tvs 115.8 ± 0.20c 55.4 ± 0.24b 102.2 ± 0.20c 49.4 ± 0.24b Hot extract + Tk 116.4 ± 0.24c 55.6 ± 0.24b 102.8 ± 0.20c 49.6 ± 0.24b Cool extract 93.6 ± 0.24g 44.4 ± 0.24d 88.6 ± 0.24h 35.0 ± 0.44g Hot extract 92.6 ± 0.24h 43.4 ± 0.24e 90.6 ± 0.24g 37.6 ± 0.24f Control 78.4 ± 0.24l 33.8 ± 0.37j 60.4 ± 0.24k 24.2 ± 0.20k The presented data are the mean ± standard errors, and the letters show significance at P ≤ 0.05 Elshahawy and El-Sayed Egyptian Journal of Biological Pest Control (2018) 28:48 Page 9 of 11 of wheat (Triticum aestivum L.) var. Giza 69 was investi- 4369.3, 2227.0 and 2229.4, and 348.0 and 352.1, respect- gated by Shabaan (2010). They reported that 50% (v/v) ively, compared to the control, being 1165.7, 893.1, and microalgae extracts foliar spray, after 25 days of sowing, 195.0 g. In contrast, plants treated with cool extract of increased plant growth and grain weight by 140 and 40%, C. vulgaris alone produced the lowest yield compared to respectively. The present study showed that the addition the other treatments (Table 6). of C. vulgaris water extracts to the culture medium or soil The same trend was observed in regard to 100-kernel increased the fresh and dry weight of maize seedlings. weight (Table 6). The highest weight was recorded in the Abd El-Motty et al. (2010)reported thatspraying of 2% case of combination treatments of Trichoderma spp. with C. algae combined with 0.2% yeast on Keitte mango trees vulgaris extracts. The lowest 100 grain weight was obtained once at full bloom had improved nitrogen, potassium, and by the control treatment (28.34 g). Data in Table 6 also indi- boron contents in the leaves. In this respect, Taha and cated insignificant differences among treatments concerning Youssef (2015) reported a significant increase of growth ear parameters of ear diameter, ear length, no. of rows per mass as well as content of phosphorous, potassium, and ear, and no. of kernels per row. All treatments increased ear chlorophyll of maize plants grown in soil treated with parameters compared to check plants. Combined treatments green microalgae strains of Chlorella. of Trichoderma spp. with C. vulgaris extracts caused the highest ear parameters. Microalgae extracts containing Field experiment many nutrients as N, P, Ca, K, S, and Mg, as well as some Following the greenhouse experiments, field experiments trace elements as Fe, Zn, Mn, Mo, Co, and Cu and some were the advanced confirmation for the aim of this work. growth regulators, vitamins, and polyamines, were applied Data in Tables 6 and 7 showed that seed + soil treatment to stimulate vegetative growth, nutritional levels, yield, and with Trichoderma spp. either alone or in combination fruit quality of different orchard as well as vineyards (Abd with C. vulgaris extracts significantly improved crop El-Migeed et al. 2004 and Spinelli et al. 2009). production and ear characters compared to check plants. Moreover, combination of Trichoderma spp. with C. vul- Conclusions garis extracts caused markedly an increase in maize yield Trichoderma spp. are one of the proven biological con- parameters compared to the individual treatments. All trol agents. In the present study, the antagonism of Tri- treatments increased the yield of maize plants. The choderma strains in combination with C. vulgaris treatment of T. virens in combination with cool and hot extracts increased the efficiency of controlling the maize extracts of C. vulgaris caused the highest of ears, weight late wilt disease. Treatments also increased maize plant of grains, and weight of grains/plant, being 4338.0 and growth and yield. It is suggested that extracellular Table 6 Effect of Trichoderma spp. alone or in combination with Chlorella vulgaris extract on yield of maize plants grown under field conditions Treatment Maize plants grown under field conditions Av. weight of ears/plot (g) Av. weight of grains/plot (g) Av. weight of grains/plant (g) T. harzianum (Th) 2780.0 ± 14.4f 1796.7 ± 2.66g 227.0 ± 1.77de T. viride (Tv) 2716.0 ± 10.5f 1688.6 ± 0.32h 208.0 ± 1.086e T. virens (Tvs) 3243.3 ± 12.3d 2059.7 ± 3.80c 235.4 ± 1.70cde T. koningii (Tk) 3068.0 ± 13.0e 2046.9 ± 0.85d 226.8 ± 2.88de Cool extract + Th 3510.0 ± 8.6c 1934.3 ± 6.28e 310.4 ± 32.67ab Cool extract + Tv 3495.0 ± 22.9c 1830.4 ± 3.35f 261.3 ± 25.43bcde Cool extract + Tvs 4338.0 ± 10.3a 2227.0 ± 1.26ab 348.0 ± 38.19a Cool extract + Tk 4046.0 ± 19.6b 2220.2 ± 0.29b 327.4 ± 37.52ab Hot extract + Th 3520.0 ± 13.2c 1929.0 ± 3.95e 302.8 ± 34.99abc Hot extract + Tv 3490.0 ± 21.7c 1827.0 ± 5.59f 296.6 ± 32.12abcd Hot extract + Tvs 4369.3 ± 27.2a 2229.4 ± 2.57a 352.1 ± 38.86a Hot extract + Tk 4040.3 ± 5.6b 2224.2 ± 1.62ab 311.6 ± 35.74ab Cool extract 2003.3 ± 26.0h 1500.6 ± 0.36i 208.0 ± 0.68e Hot extract 2185.3 ± 14.6g 1503.0 ± 0.61i 205.6 ± 0.16e Control 1165.7 ± 70.3i 893.1 ± 5.97j 195.0 ± 1.11e The presented data are the mean ± standard errors, and the letters show significance at P ≤ 0.05 Elshahawy and El-Sayed Egyptian Journal of Biological Pest Control (2018) 28:48 Page 10 of 11 Table 7 Effect of Trichoderma spp. alone or in combination with Chlorella vulgaris extract on average parameters of yield component of maize plants grown under field conditions Treatment Average parameters of yield component of maize plants grown under field conditions Av. ear length (cm) Av. ear diameter (cm) No. of row/ear No. of kernel/row 100 kernel weight (g) T. harzianum (Th) 22.59 ± 0.05e 4.11 ± 0.03e 12.8 ± 0.33abc 40.2 ± 0.13g 32.29 ± 0.07fg T. viride (Tv) 22.31 ± 0.08f 4.26 ± 0.03 cd 12.4 ± 0.26c 40.7 ± 0.15fg 32.14 ± 0.09g T. virens (Tvs) 22.58 ± 0.10e 4.28 ± 0.03bcd 12.4 ± 0.26c 41.6 ± 0.16e 32.78 ± 0.08defg T. koningii (Tk) 22.79 ± 0.02cd 4.35 ± 0.01a 13.0 ± 0.33abc 41.0 ± 0.25ef 32.53 ± 0.18efg Cool extract + Th 22.99 ± 0.01a 4.35 ± 0.01a 13.2 ± 0.32abc 44.6 ± 0.22bc 33.47 ± 0.13bcd Cool extract + Tv 22.90 ± 0.02abc 4.24 ± 0.01d 13.0 ± 0.33abc 43.5 ± 0.16d 33.32 ± 0.14cde Cool extract + Tvs 22.85 ± 0.02bc 4.36 ± 0.01a 13.6 ± 0.26a 45.0 ± 0.25ab 33.52 ± 0.17abcd Cool extract + Tk 22.94 ± 0.02ab 4.36 ± 0.01a 13.4 ± 0.30ab 45.0 ± 0.21ab 33.56 ± 0.15abcd Hot extract + Th 22.94 ± 0.02ab 4.24 ± 0.01d 13.0 ± 0.33abc 44.7 ± 0.26bc 33.76 ± 0.06abc Hot extract + Tv 22.85 ± 0.02bc 4.24 ± 0.01d 12.8 ± 0.32abc 44.3 ± 0.15c 33.02 ± 0.09cdef Hot extract + Tvs 22.93 ± 0.03ab 4.34 ± 0.03ab 13.6 ± 0.26a 45.5 ± 0.22a 34.23 ± 0.01ab Hot extract + Tk 22.97 ± 0.02ab 4.32 ± 0.02abc 12.6 ± 0.30bc 45.2 ± 0.24ab 34.35 ± 0.12a Cool extract 22.66 ± 0.03de 4.24 ± 0.01d 12.4 ± 0.26c 40.4 ± 0.16fg 30.19 ± 0.11h Hot extract 22.78 ± 0.03cd 4.26 ± 0.01cd 12.4 ± 0.26c 40.5 ± 0.16fg 29.66 ± 0.39h Control 22.37 ± 0.08f 3.86 ± 0.01f 10.4 ± 0.26d 33.6 ± 0.45h 28.34 ± 1.04i The presented data are the mean ± standard errors, and the letters show significance at P ≤ 0.05 saccharides content of C. vulgaris extracts enhanced the Alhanshoul AM (2015) Studies on maize late wilt disease caused by Cephalosporium maydis. Ph.D. thesis. Fac. Agric., Cairo Univ., Egypt, p 166 growth and adhesion of Trichoderma spp. which pro- Ali M (2000) Diversity in isolates of Cephalosporium maydis the causal of late wilt moted plant growth through increasing antifungal of maize in Egypt. M.Sc. Thesis. Fac. Agric., Cairo Univ., Egypt, p 120 activity. Anonymous (2017) Bulletin of the agricultural statistics. Ministry of Agric. and Land Reclamation, Egypt, p 215 Acknowledgements Ashour AM, Sabet KA, El-Shabrawy EM, Alhanshoul AM (2013) Control of maize The authors wish to thank the Department of Corn Diseases and Sugar late wilt and enhancing plant growth parameters using rhizobacteria and Crops Research, Plant Pathology Research Institute, Agriculture Research organic compounds. Egypt J Phytopath 41(2):187–207 Center, Giza, Egypt, for providing the field experiment. Bell DK, Wells HD, Markham CR (1982) In vitro antagonism of Trichoderma species against six fungal plant pathogens. 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Egyptian Journal of Biological Pest Control – Springer Journals
Published: May 29, 2018
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