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
Testing the antagonistic activities of Trichoderma spp. which uses either alone or in combination with C. vulgaris extracts against C. maydis was carried out. In the case of Trichoderma spp. alone, the inhibitor effect of T. harzianum, T. koningii, T. viride, and T. virens against the growth of the most virulent isolate of C. maydis (isolate Cm3) was studied, using the method described by Bell et al. (1982). Petri plate containing PDA medium supplemented with 0.2% yeast extract was inoculated on one side with a 5-mm mycelial disc from a 7-day-old culture of the tested Trichoderma spp. The opposite side was inoculated with a disc of C. maydis, and the plates were incubated at 28 ± 2 °C. Plates inoculated with a disc of C. maydis alone were used as control. Four replicate plates were made for each test fungus as well as for the control. Colony radius of C. maydis was recorded when the control plates reached full growth. On the other hand, the effect of C. vulgaris water extracts on the antagonistic activity of Trichoderma spp. against C. maydis was carried out, using PDA plates amended with each of cool or hot extracts. Ten milliliters of each extract was filtered through a sterile 0.22-μm Millipore filter directly into 190 ml molten PDA. The medium was poured into sterile Petri plates and cooled at room temperature. The amended plates were used for dual culture test described before. Plates amended with cool extract, hot extract, and sterile distilled water and inoculated with a disc of C. maydis by itself were used as control. Four replicate plates were used for each treatment as well as for controls. Colony radius of C. maydis was recorded when the control plates reached full growth. The reduction in the growth of C. maydis was calculated, using the following formula:
$$ \mathrm{Growth}\ \mathrm{reduction}\ \left(\%\right)=\left[\left(C-T\right)/C\right]\times 100. $$
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.