2.1 Local of study and sampling
The research was conducted in laboratory conditions at the Universidade Federal do Tocantins (UFT), Gurupi-TO, Brazil (11º43'45"S, 49º04'07" W and 278 m). Samples of the "Sempre Verde" cultivar were collected from commercial cowpea seeds collected in July 2019 from family farmers of the city of Pedro Afonso, Latitude: -8.9715, Longitude: -48.17548° 58′ 17″ Sul, 48° 10′ 31″ Oeste. The seeds were stored in plastic bottles and taken to the Laboratory of Symbiosis and Insects-Microorganisms at the Universidade Federal of Tocantins (UFT), Gurupi campus.
2.2 Health Assessment of cowpea seeds
The blotter test method detected the pathogen fungi associated with the cowpea seeds. The health assessment of the seeds was carried out in two assays. The first one assessed the incidence of fungi in the whole seeds with two treatments, with asepsis and without asepsis. The asepsis treatment consisted of surface disinfection of the seeds with alcohol 70% for two minutes, followed by 1% sodium hypochlorite for two minutes. Furthermore, subsequently washed three times with sterile distilled water.
Four 400 seeds were distributed in Gerbox plastic boxes, 25 seeds per dish, with the germitest paper moistened with autoclaved distilled water. After 24 hours, the box was placed in a refrigerator to kill the embryo of the seeds. After 24 hours, the box was transferred to a climatic chamber at 25 ± 2°C with a photoperiod of 12 hours to observe the growth of seed-borne fungi daily for 15 days.
The second assay assessed the fungal incidence in peeled seeds and the integument with asepsis and without. The asepsis was performed through the same method described above. The seeds were watered and placed in Gerbox plastic boxes with germitest paper moistened with autoclaved distilled water. After 24h, the integument was removed using a disinfected blade. Then, the integuments and the peeled seeds were distributed in different treatments; 25 of each per gerbox totalled 400 integuments and 400 peeled seeds.
The box was transferred to a climatic chamber to observe the growth of seed-borne fungi daily for 15 days. The incidence of fungi in the seeds was expressed in percentage. With morphological observation by a microscope, initially, the fungi were identified at the genus level with the help of the specialized bibliography (Ellis 1971; Barnett & Hunter 1972). The potential phytopathogenic isolates observed were plated in dishes with potato dextrose agar (PDA) culture medium in BOD at 25 ± 2°C with a photoperiod of 12 hours for further identification and assays.
2.3 Pathogenicity of the seed-borne fungi isolates
Koch's postulates were performed to confirm the pathogenicity of the fungi isolated from the seeds in the health tests. Cowpea seeds were disinfected with the asepsis method described above and sowed in a polyethene bag filled with autoclaved commercial substrate. The seedlings were wet once a day, and the inoculation was done after 15 days. The inoculation was done by applying a 6 mm diameter disc of mycelium of each isolate on the leaves using a sterilized blade (dos Santos et al. 2022).
A completely randomized design was used, with four treatments (fungi isolates) and three replicates. Each repetition consisted of one plant. The control consisted of four healthy, non-inoculated plants. After the inoculation, the seedlings were covered with a transparent plastic bag and kept under 25°C in a chamber for 48h to maintain the humidity and facilitate the sporulation and the infection of the isolates. After 48h, the inoculated seedlings were transferred to the greenhouse until the appearance of the pathogen symptoms for evaluated.
A process of isolation was performed from the lesions observed in the leaves to confirm the pathogen identity. The pathogens were incubated in plate dishes with PDA and purified to obtain a pure culture and then reinoculated in healthy seedlings to confirm the pathogenicity of the isolates. Isolates confirmed as pathogens were submitted to molecular identification and used in other bioassays.
2.4 Molecular identification of the phytopathogens isolated from cowpea seed
Three potential pathogenic isolates were identified by analyzing the internal transcribed spacer (ITS) regions of their nuclear ribosomal DNA (rDNA). The fungi gDNA extraction, PCR amplification (ITS1 F: 5’- TCCGTAGGTGAACCTGCGG -3’ ITS4 R: 5’- TCCTCCGCTTATTGATATGC-3), Sanger sequencing, and assembly were performed by DSMA (http://www.dsma.com.br/). The extraction of DNA was carried out using the Phenol-chloroform method. Polymerase Chain Reaction amplified the ITS rDNA primers. The PCR amplification products were purified with Microcon™ filters (Millipore). Single-pass sequencing was performed in Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems).
The results of the sequencing were compared with sequences published in databases (NCBI, RDP) (GenBank (http://blast.ncbi.nlm.nih.gov). The most similar GenBank sequences to our results were used to build phylogenetic trees. The sequences were aligned using CodonCodeAligner V4.2.7 (LI-COR®), and the phylogenetic analysis was performed using the MEGA X software.
2.5 Antagonistic effects of Beauveria bassiana against phytopathogen isolates in vitro
According to the methodology of paired cultures (Campanile et al. 2007), B. bassiana was compared against the phytopathogen isolates to assess its potential for antagonism. Mycelial discs of 5 mm of the fungi were inoculated in the Petri dishes containing the potato dextrose agar (PDA) medium at a 3 cm distance to a disc of equal size to the phytopathogen isolates. The plates were kept at 28°C in BOD for ten days. Then, the assessment was performed by measuring the diameter of colonies in two diametrically opposite directions using a pachymeter. The percentage of mycelial growth inhibition (PIC) was calculated using the formula PIC: ⌊(C-T) /C⌋x100, where C is the control diameter, and T is the treatment diameter.
Considering the difference between the slow growth of B. bassiana and the rapid growth of pathogens, the assays were performed twice on the first day of inoculation of B. bassiana and the second carried out three days after.
2.6 Antagonistic effects of Beauveria bassiana against phytopathogens isolates in vivo
The effect of B. bassiana against phytopathogens isolates was evaluated. Seeds of cowpea were first surface-sterilized with the asepsis method. The seeds were planted in pots filled with autoclaved soil. Three seeds were planted per container. One week after germination, the least vigorous seedling in each pot was discarded. The plants were kept in laboratory conditions with artificial light, 180 μmol m-2 s-1, 75% relative humidity, at 27 °C and 12 h of photoperiod, and wet when necessary.
The inoculation of the pathogen isolates was carried out 15 after the planting by applying a 6 mm diameter disc of mycelium of each isolate on the leaves, using a sterilized blade (dos Santos et al. 2022). Inoculated seedlings were covered with a transparent plastic bag for 48h in a randomized block design. The treatment was B. bassiana with six replications with three blocks, each isolate representing a block. The inoculation was carried out in 6 leaflets per plant, each representing a repetition. Each isolate received a control: the plant with the phytopathogen isolate; the treatment was the inoculation of B. bassiana. Seedlings did positive control without any fungi inoculation to compare the symptoms of fungi that the seeds could transmit.
The inoculation of the B. bassiana was done seven days after the inoculation of the pathogens when the lesions of the pathogens appeared using the same method described above. The control effects of B. bassiana were assessed by observing the inhibition of the spread of the pathogens or the disappearance of the symptoms compared with the control (Bock et al. 2021).
2.4 Statistical analysis
The results obtained in the antagonism assays (growth of inhibition and diminution of symptoms) were submitted to analysis of variance (ANOVA) by the Sisvar software (Ferreira 2011).