Maximizing the efficacy of Trichoderma to control Cephalosporium maydis, causing maize late wilt disease, using freshwater microalgae extracts
- Ibrahim E. Elshahawy1Email author and
- Abo El-Khair B. El-Sayed2
https://doi.org/10.1186/s41938-018-0052-1
© The Author(s) 2018
Received: 12 March 2018
Accepted: 16 May 2018
Published: 29 May 2018
Abstract
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
Background
Maize, Zea mays L., is one of the most important cereal crops worldwide. In Egypt, the cultivated maize area reached about 88,000 ha that yielded almost 7.2 million metric tons of grains (Anonymous 2017). Black bundle disease or late wilt, caused by Cephalosporium maydis, is one of the main economical and distributed maize diseases in Egypt (Samra et al. 1963). This disease appears during tasseling as a rapid wilting of the lower leaves and develops to hollow and shrunken stalks with a dark yellow-to-brown or black-stained pith (El-Shafey and Claflin 1999). The pathogen is a soil-borne vascular wilt disease that enters tissue of the root and colonizes the xylem (Sabet et al. 1970). Less commonly, this pathogen can be seed-borne (E1-Shafey et al. 1976) and may irregularly cause decay of seed or pre-emergence damping-off under heavy inoculum pressure (Sabet et al. 1970). This fungus duplicates asexually and has not been in perfect stage (Saleh and Leslie 2004). Large economic losses have been reported in Egypt by late wilt disease. In susceptible varieties, the disease affected 70% of the plants decreasing the grain yield by 40% (Labib et al. 1975). Breeding of resistant varieties of maize is the most effective method for controlling this disease (El-Shafey et al. 1988). Various bacteria and actinomycetes have been evaluated as biocontrol agents against late wilt disease (El-Mehalowy et al. 2004 and Ashour et al. 2013). Little information has been cited in the literature on the efficiency of Trichoderma spp. against late wilt disease. Trichoderma spp. isolated from Egyptian soil were used as a biocontrol for Colletotrichum dracaenophilum and Fusarium proliferatum, based on results of laboratory trials (Morsy and Elshahawy 2016 and Elshahawy et al. 2017a). It reduced disease caused by the soil-borne fungus Stromatinia cepivora (Berk.) and induced plant resistance in onion plants when applied to soil (Elshahawy et al. 2017b).
The microalgae, Chlorella vulgaris known as freshwater algae, is one of the most remarkable green microalgae. There are several applications and potential benefits of this microalga such as biofuels, human nutrition, animal feed, wastewater treatment, and agrochemical applications (Safi et al. 2014). C. vulgaris contains high amounts of micro- and macronutrients, proteins, and carbohydrates (Wake et al. 1992). It is used as bio-fertilizer and soil conditioner in agriculture systems (Song et al. 2005). Algal extract can be partially substituting micronutrient foliar fertilizers and best to be complementary portion of the spray solution (Shabaan 2010). Soil fertility can be improved by entrapping some rhizosphere bacteria with Chlorella (Raposo and Morais 2011). Newly, the consortium of C. vulgaris, Azotobacter sp., and Anabaena variabilis was found to increase germination and plant growth of rice, and it is suggested as a bio-fertilizer and a bio-stimulator for crops as reported by Zayadan et al. (2014).
The present study was conducted to evaluate the efficiency of Trichoderma species either alone or mixed with the C. vulgaris extracts on the incidence of maize with late wilt under greenhouse and field conditions.
Materials and methods
Experimental site
This study was carried out at the Agriculture and Biological Division, National Research Centre (NRC), as well as within a disease nursery field located at Gharbia Governorate, Egypt, during the 2016 growing season.
Freshwater microalgae, Chlorella vulgaris, and preparation of extracts
C. vulgaris was isolated from freshwater Nile River at Cairo Governorate, Egypt (El-Sayed et al. 2001). This strain was massively produced at Algal Biotechnology Unit, National Research Centre, Giza, Egypt. The cultivation was performed, using a 1200-l open-plate photobioreactor. Microalgae nutrition was performed as described by El-Sayed et al. (2015). Grown culture was concentrated and dewatered by gravity. Purification of the obtained biomass was performed by a series of precipitation by cooling centrifuge and washing it using tap water. This procedure was repeated several times to remove any excess of nutrients and mineral elements. The obtained biomass was dried at 45 °C within a circulated oven and then ground to a fine powder (Hassan et al. 2015).
Hot (at 70 °C) and cool water extracts were produced by soaking 10% of microalgae biomass with distilled water and solicited using ultrasonic homogenizer. After homogenization, the extracted materials were obtained by filtration through filter paper (Whatman no. 1). The extracts were freeze-dried and sieved in a refrigerator until used. Total sugars were determined according to Dubois et al. (1956). Polysaccharides were determined in extracts. Firstly, freeze-dried extracts were sequentially treated by petroleum ether and chloroform to remove oiled materials. Absolute ethanol was used to precipitate polysaccharides. Forty milliliters of absolute ethanol was added gradually to 10 ml of water extracts (1:20 w/v). The mixtures were left overnight into the refrigerator and then centrifuged (5500 rpm for 10 min). The precipitated polysaccharides were dried using a freeze drier and determined by gas-liquid chromatography (GLC).
Trichoderma species
Four Trichoderma species, viz., T. harzianum, T. koningii, T. viride, and T. virens, were obtained from Plant Pathology Department, NRC, Egypt. The Trichoderma species were isolated from Egyptian soil, identified, and evaluated for their efficiency in previous study (Elshahawy et al. 2016).
C. maydis isolates
Maize plant samples, showing typical late wilt symptoms, were collected from naturally infected fields located at Gharbia Governorate, Egypt. Isolation of C. maydis was carried out according to Samra et al. (1963). Stems of diseased maize plants were cut into small pieces, and the surface was disinfected with 0.5% sodium hypochlorite for 3 min and then washed thoroughly with sterilized water. The disinfected stem pieces were dried between folds of sterile filter papers, then plated onto potato dextrose agar (PDA) medium supplemented with 0.2% yeast extract and incubated at 28 ± 2 °C for 72 h. Hyphal tip isolation technique was employed to obtain the fungus isolation in pure cultures. C. maydis was identified according to morphological and cultural features using the descriptions of Samra et al. (1963) and Ainsworth and James (1971). Five isolates of C. maydis were obtained from diseased maize plants and kept at 4 °C for further studies.
Inoculum preparation and determination of pathogenicity
The isolates of C. maydis were grown into 250 ml potato dextrose broth medium supplemented with 0.2% yeast extract in 500 ml Erlenmeyer flasks. After sterilization, flasks were inoculated with each of the different isolates of C. maydis and then incubated at 28 ± 2 °C for 2 weeks. The flasks were thoroughly shaken, and about 20 ml of the suspension was poured into 1-l glass bottles containing wet autoclaved crushed grain sorghum up to two thirds of its capacity. The inoculated glass bottles were then kept at 28 ± 2 °C for 4 weeks. Pathogenicity test of the obtained isolates of C. maydis was conducted on a susceptible maize cultivar Baladi. Disinfested grain seeds were planted in pots (30 cm in diameter) containing autoclaved clay loam soil (6 kg/pot), infested with the inoculum of different isolates. Seed disinfestations were carried out by soaking seeds in 5% sodium hypochlorite solution for 3 min and rinsed in sterile water. Pots and soil were treated 2 weeks before planting by autoclaving the soil and soaking the pots in 7% formalin solution for 3–5 min. Soil infestation was carried out 7 days before planting by mixing 180 g of inoculum to the soil in every pot and mixed thoroughly to ensure equal distribution of fungal propagates, followed by irrigation. Each pot was seeded with eight grain seeds of the Baladi cv., and plants were thinned to three plants per pot. Six pots were used for each isolate, and a non-inoculated treatment 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. Percentage of dead plants due to late wilt infection was calculated 80 days after planting. Disease symptoms began to appear approximately 60 days after sowing. Pots were examined at weekly intervals thereafter and symptomatic plants removed when they were identified. Fungal isolates were recovered from internodes of symptomatic plants to demonstrate Koch’s postulates. Among the tested isolates, the highest aggressive isolate was selected and used throughout the present study. The maize plants were harvested at 80-day age by mulching the plants from the pots. The length of plants and their dry weight were determined. The harvested plants were dried at 70 °C till constant weight, and the dry weight per plant was recorded.
Laboratory experiments
Antagonistic activity tests
where C is the average linear growth of C. maydis in control and T is the average linear growth of C. maydis in biocontrol agent treatment.
Development of Trichoderma–C. vulgaris extract formulation
Trichoderma spp. propagules
Trichoderma harzianum, T. koningii, T. viride, and T. virens were grown on a PDA medium at 25 ± 2 °C for 10 days. Afterwards, the mycelium with the spores was scraped from Petri plates and mixed with sterilized distilled water (20 ml/plate) in a blender. The suspension was adjusted by a hemocytometer slide to 108 propagates/ml.
Preparation of C. vulgaris extracts
Each of cool or hot extracts of C. vulgaris was prepared individually. Two hundred and fifty milliliters of each extract was filtered through a sterile 0.22-μm Millipore filter directly into a 500-ml sterile conical flask.
Incorporation of Trichoderma spp. to C. vulgaris extracts
Propagule suspension (108 propagates/ml) of each of Trichoderma spp. were individually incorporated into sterilized C. vulgaris extracts under aseptic conditions at the rate of 10 ml of suspension per 90 ml extract and thoroughly shacked on a rotatory shaker at 70 rpm for 6 h. Each Trichoderma–C. vulgaris extract was first stored at room temperature for 3 days to increase the initial population of Trichoderma spp., and then, they were applied.
Population dynamics of Trichoderma spp. on C. vulgaris extracts
The viability of Trichoderma spp. in C. vulgaris extracts was determined at 3, 60, 120, 180, 240, 300, and 360 days after storage (DAS) of room temperature (27 ± 2 °C). For the study of the potentiality of 7 °C storage conditions on the viability of the Trichoderma spp. in C. vulgaris extracts, they were first stored at room temperature for 3 days to increase the initial population of Trichoderma spp. Initial determination of population of Trichoderma spp. was made at 3 DAS at room temperature, and later samples were made at 60, 120, 180, 240, 300, and 360 DAS at 7 °C. Serial dilutions of formulation samples were used to determine the number of Trichoderma spp. propagules found on C. vulgaris extracts by the plate count technique using selective media (Johnson et al. 1960). Thus, the blended 1 ml of formulation was transferred to bottles containing 99 ml of sterilized distilled water under aseptic conditions. The bottles were shaken using a mechanical shaker for 15 min. Serial dilutions of fresh suspension were prepared for each Trichoderma spp. in C. vulgaris extract sample under sterile conditions. A portion of 1.0 ml formulation suspension from the dilution 10−4 was transferred to four sterile Petri plates. Rose Bengal streptomycin-selective medium was used for growing Trichoderma spp. colonies after 4 days of incubation at 25 ± 2 °C (Metcalf 1997). This medium consisted of 2.0 g of (NH4)2SO4, 4.0 g of KH2PO4, 6.0 g of Na2HPO4, 0.2 g of Fe·SO47H2O, 1 mg of CaCl2, 10 μg of H3BO3, 10 μg of MnSO4, 70 μg of Zn SO4, 1 l of distilled water, 20 g agar, and 5 g of cellulose powder (Sigma), adjusted to pH 4.0 before autoclaving. After the medium cooled to 70 °C, 0.05 g of streptomycin sulfate and 0.016 g of rose Bengal were added.
Greenhouse experiments
A pot experiment was conducted to evaluate the influence of Trichoderma spp. treatments alone or formulated on C. vulgaris extracts on the incidence of maize late wilt as well as on growth parameters of maize plant in soil infected and non-infected with late wilt pathogen. The experiment was conducted in the summer season of 2016 at the greenhouse of Plant Nutrition Department, Agriculture and Biological Division, National Research Centre, Egypt. The experiment was carried out in a randomized complete block design with four replicates. The most virulent isolate of C. maydis (isolate Cm3) was used. Seed disinfections were carried out by soaking seeds in 5% sodium hypochlorite solution for 3 min, rinsed in sterile water. Pots (30 cm in diameter) and soil were treated 2 weeks before planting by autoclaving the soil and soaking the pots in 7% formalin solution for 3–5 min. Soil infestation was carried out 7 days before planting by mixing 180 g of C. maydis inoculum to the soil in every pot (6 kg soil/pot), followed by irrigation. Disinfected maize grains (Baladi cv.) were soaked in each treatment at the rate of 100 ml/100 grain in 250-ml Erlenmeyer flasks. Control of grains was soaked in sterile distilled water only. Few drops of Tween-80 were added to improve adhesive. Flasks were incubated at 25 °C on a rotary shaker at 70 rpm for 6 h to allow treatment materials to adhere to seeds. After incubation, excess inoculum was removed and grains were left to air-dry for 30 min at room temperature and then immediately planted in the infected and/or un-infected potted soil (Ashour et al. 2013). Each pot was seeded with eight grain seeds, and the plants were thinned to three plants. The abovementioned treatments were applied to soil in the pots with irrigation water at three equal doses (30 ml per pot) each 10 days. Six pots were used for each treatment as well as control. Nitrogen fertilizer in the form of urea (46% N) was added at the rate of 500 mg N/kg soil, 30 days after planting, and the plants were irrigated when necessary.
The following treatments were used in soil infected and non-infected with late wilt pathogen: (1): T. harzianum (10 × 104 propagates/ml sterile distilled water), (2): T. koningii (10 × 104 propagates/ml sterile distilled water), (3): T. viride (10 × 104 propagates/ml sterile distilled water), (4): T. virens (10 × 104 propagates/ml sterile distilled water), (5): T. harzianum (10 × 104 propagates/ml cool water extract of C. vulgaris), (6): T. koningii (10 × 104 propagates/ml cool water extract of C. vulgaris), (7): T. viride (10 × 104 propagates/ml cool water extract of C. vulgaris), (8): T. virens (10 × 104 propagates/ml cool water extract of C. vulgaris), (9): T. harzianum (10 × 104 propagates/ml hot water extract of C. vulgaris), (10): T. koningii (10 × 104 propagates/ml hot water extract of C. vulgaris), (11): T. viride (10 × 104 propagates/ml hot water extract of C. vulgaris), (12): T. virens (10 × 104 propagates/ml hot water extract of C. vulgaris), (13): Cooled water extract of C. vulgaris, (14): Heat water extract of C. vulgaris, (15): Control.
Percentage of dead plants due to late wilt infection was recorded 80 days after planting. Vegetative growth parameters, i.e., plant height and dry weight, were also recorded as previously described.
Field experiments
The effect of Trichoderma spp. treatments alone or formulated on C. vulgaris extracts on the incidence of maize late wilt as well as on yield of maize plant was studied under field conditions in a disease nursery at Gemmiza Research Station, Plant Pathology Research Institute, Agriculture Research Center, Gharbia Governorate, Egypt, during the 2016 growing season. This nursery was infested artificially with the four clonal lineages of C. maydis found in Egypt that causes late wilt of maize and commonly used in Egyptian maize breeding programs (Zeller et al. 2002). Maize grains cv. Baladi were used in this study. The abovementioned treatments in greenhouse were involved in field experiments. Disinfected maize grains (Baladi cv.) were soaked in each treatment at the rate of 100 ml/100 grain. Control grains were soaked in sterile distilled water only. Randomized complete block arrangement in three replicate plots was used. Each replicate included three ridges of 4.5-m length and 0.7-m width for each ridge, i.e., the experimental plot area was 3.15 m2. Thirteen maize plants for each treatment were used in each replicate. Grains were sown in holes (five holes/ridge with three grains/hole); thereafter, they were thinned to one plant/hole. The abovementioned treatments were also applied before irrigation with water at three equal doses (10 ml per hole) each 15 days. Irrigation, recommended fertilizer levels, and agronomical practices were used as usual. Disease incidence of late wilt as infection percentage was recorded 110 days after sowing. Quantitative maize yield and qualitative maize yield, i.e., ear length, ear diameter, no. of rows per ear, no. of kernels per row, no. of kernels per ear, and 100-kernel weight, were evaluated during harvest period.
Statistical analysis
Statistical analysis of data was conducted, using SPSS software version 14.0. Percent data of disease incidence were statistically analyzed after arcsine square root transformation; however, untransformed data are presented. Analysis of variance was determined, and the mean values were compared by Duncan’s multiple range test at P < 0.05.
Results and discussion
Chemical analysis of Chlorella extract
Intercellular saccharide content (% dw from total carbohydrates) of Chlorella vulgaris cool and hot extract
Saccharide | % content (dry weight from total carbohydrates) | |
|---|---|---|
Cool | Hot | |
Galactose | 25.5 | 26.30 |
Mannose | 16.3 | 11.90 |
Rhamnose | 14.5 | 18.20 |
Glucose | 13.4 | 10.60 |
Arabinose | 09.7 | 10.70 |
Xylose | 06.6 | 08.90 |
Fructose | 02.9 | 02.96 |
Ribose | 02.8 | 02.03 |
C. maydis isolation
Virulence of Cephalosporium maydis isolates on maize cv. Baladi under greenhouse conditions
Isolate | Disease incidence (%) | Plant’s vigor | |
|---|---|---|---|
Plant height (cm) | Plant dry weight (g) | ||
C. maydis (Cm1) | 67.2 ± 0.37c | 66.2 ± 0.37bc | 29.2 ± 0.48b |
C. maydis (Cm2) | 65.0 ± 1.00d | 64.6 ± 0.40c | 27.0 ± 0.31c |
C. maydis (Cm3) | 78.2 ± 0.37a | 59.4 ± 0.60d | 23.6 ± 0.50d |
C. maydis (Cm4) | 74.4 ± 1.16b | 66.8 ± 0.37b | 28.0 ± 0.31bc |
C. maydis (Cm5) | 67.6 ± 0.60c | 66.6 ± 0.24bc | 26.8 ± 0.37c |
Control | 00.0 | 83.4 ± 1.40a | 34.8 ± 0.20a |
Antagonistic activity of Trichoderma spp. and C. vulgaris extracts against C. maydis
Antagonistic activity of Trichoderma spp. alone or in combination with Chlorella vulgaris extract against the linear growth of Cephalosporium maydis
Treatment | Linear growth and growth reduction of C. maydis | |
|---|---|---|
Linear growth (cm) | Reduction (%) | |
T. harzianum (Th) | 3.30 ± 0.20d | 63.3 |
T. viride (Tv) | 4.50 ± 0.15c | 50.0 |
T. virens (Tvs) | 2.20 ± 0.12f | 75.6 |
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 |
Cool extract + Tvs | 1.12 ± 0.04h | 87.6 |
Cool extract + Tk | 1.28 ± 0.03h | 85.8 |
Hot extract + Th | 1.80 ± 0.12g | 80.0 |
Hot extract + Tv | 3.56 ± 0.16d | 60.4 |
Hot extract + Tvs | 1.08 ± 0.04h | 88.0 |
Hot extract + Tk | 1.18 ± 0.04h | 86.9 |
Cool extract | 7.88 ± 0.08b | 12.4 |
Hot extract | 7.64 ± 0.17b | 15.1 |
Control | 9.00 ± 0.00a | 00.0 |
Population dynamics of Trichoderma spp. on C. vulgaris extracts
The results of the effect of cool and heat extracts of C. vulgaris-based liquid formulations on the population dynamics of Trichoderma spp. at room temperature indicated that the algae extracts supported the highest population of Trichoderma spp. during the DAS sampled (data not shown). The population of Trichoderma spp. found on cool and heat extracts of C. vulgaris followed a fluctuating trend with the DAS sampled. The initial population (3 days) of Trichoderma spp. was increased at 60 DAS. At 120 DAS, the population recovery in all the Trichoderma spp. was significantly increased. Thereafter, during the period of 180 to 300 DAS, the population of Trichoderma spp. was declined progressively, and at 360 DAS, a significant reduction of more than four- to ninefold was recorded. The results of the population dynamics of Trichoderma spp. in extracts of C. vulgaris, when the liquid formulations were stored at 7 °C, showed that there was a slow and progressive decline of the antagonist populations in the bioformulations from 120 DAS to 180, 240, 300, and 360 DAS. Still, the population recovered was much greater as compared to that recorded during the same period under room temperature storage conditions. These 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 (data not shown).
Efficiency of treatments on maize late wilt disease
Effect of Trichoderma spp. alone or in combination with Chlorella vulgaris extract on the incidence of late wilt of maize grown under greenhouse and field conditions
Treatment | Late wilt incidence (%) | |
|---|---|---|
Greenhouse experiment | Field experiment | |
T. harzianum (Th) | 45.2 ± 0.86c | 31.8 ± 0.48c |
T. viride (Tv) | 37.6 ± 0.51d | 28.6 ± 0.50d |
T. virens (Tvs) | 33.6 ± 0.24e | 23.8 ± 0.37e |
T. koningii (Tk) | 30.2 ± 0.20f | 24.6 ± 0.24e |
Cool extract + Th | 30.0 ± 0.31f | 20.8 ± 0.48f |
Cool extract + Tv | 27.6 ± 0.24g | 16.4 ± 0.40g |
Cool extract + Tvs | 23.2 ± 0.20h | 12.8 ± 0.48h |
Cool extract + Tk | 23.8 ± 0.37h | 12.4 ± 0.40h |
Hot extract + Th | 27.6 ± 0.24g | 17.6 ± 0.40g |
Hot extract + Tv | 30.0 ± 0.31f | 19.6 ± 0.74f |
Hot extract + Tvs | 21.0 ± 0.31i | 12.4 ± 0.40h |
Hot extract + Tk | 22.4 ± 0.24hi | 12.4 ± 0.40h |
Cool extract | 57.4 ± 0.81b | 46.8 ± 0.48a |
Hot extract | 57.2 ± 0.37b | 43.8 ± 0.20b |
Control | 73.4 ± 1.66a | 47.6 ± 0.24a |
In the field experiment, treatment of seed and soil with Trichoderma spp. either alone or in combination with C. vulgaris extracts showed significant reduction in maize infection with late wilt compared to check plants (47.6% infection) as presented in Table 4. The combination treatments of Trichoderma spp. with C. vulgaris extracts caused more reduction in maize late wilt compared to the individual treatments of Trichoderma spp. or C. vulgaris extracts. However, the combined treatment of C. vulgaris extract with T. virens and/or T. koningii gave the highest reduction in late wilt incidence, being 73.1 and 74.0%, respectively, in comparison to treatment with T. virens or T. koningii each alone, being 50.0 and 48.3% reduction, respectively. The lowest reduction in disease incidence was recorded by C. vulgaris extract treatments only (being 1.7 and 8.0% reduction). The antagonistic effect of Trichoderma strains against soil-borne fungi was recently emphasized by Elshahawy et al. (2017b). The reported data describe, for the first time, control of the pathogen, using Trichoderma strains in Egypt. The ability of Trichoderma strains to inhibit C. maydis pathogen is noteworthy since the suppression obtained is a result of seed + soil treatment. Addition of Trichoderma strains with C. vulgaris extracts increased their effect against C. maydis. These results suggest that C. vulgaris extracts stimulate the inhibitory activity of Trichoderma strains. This may be due to that C. vulgaris extracts are considered as absorbed agents into plants for increasing of disease and stress resistance (Abd El-Motty et al. 2010). Amendment of Trichoderma strains with C. vulgaris extracts could increase the plant protection by supporting the growth of Trichoderma strains and stimulating the useful metabolite production which may help antagonistic activity. Degani et al. (2015) reported that many plant growth promoters such as hormones, auxin (indole-3-acetic acid), and cytokinin (kinetin) were produced by higher levels, when using C. vulgaris extracts, suppressing C. maydis in culture media and in a detached root assay.
Efficiency of treatments on maize growth in greenhouse and on maize yield in field
Greenhouse experiment
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 |
Field experiment
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 |
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 same trend was observed in regard to 100-kernel weight (Table 6). The highest weight was recorded in the case of combination treatments of Trichoderma spp. with C. vulgaris extracts. The lowest 100 grain weight was obtained by the control treatment (28.34 g). Data in Table 6 also indicated insignificant differences among treatments concerning ear parameters of ear diameter, ear length, no. of rows per ear, and no. of kernels per row. All treatments increased ear parameters compared to check plants. Combined treatments of Trichoderma spp. with C. vulgaris extracts caused the highest ear parameters. Microalgae extracts containing many nutrients as N, P, Ca, K, S, and Mg, as well as some trace elements as Fe, Zn, Mn, Mo, Co, and Cu and some growth regulators, vitamins, and polyamines, were applied to stimulate vegetative growth, nutritional levels, yield, and fruit quality of different orchard as well as vineyards (Abd El-Migeed et al. 2004 and Spinelli et al. 2009).
Conclusions
Trichoderma spp. are one of the proven biological control agents. In the present study, the antagonism of Trichoderma strains in combination with C. vulgaris extracts increased the efficiency of controlling the maize late wilt disease. Treatments also increased maize plant growth and yield. It is suggested that extracellular saccharides content of C. vulgaris extracts enhanced the growth and adhesion of Trichoderma spp. which promoted plant growth through increasing antifungal activity.
Declarations
Acknowledgements
The authors wish to thank the Department of Corn Diseases and Sugar Crops Research, Plant Pathology Research Institute, Agriculture Research Center, Giza, Egypt, for providing the field experiment.
Authors’ contributions
Both authors read and approved the final manuscript.
Ethics approval
The authors declare that they have ethics approval and consent to participate.
Competing interests
The authors declare that they have no competing interests.
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