In vitro and in planta potential effect of some indigenous antagonists against Fusarium and pythiaceous species associated with peach seedlings decline

Background: The effect of Aspergillus spp. and Trichoderma harzianum isolates was evaluated against the growth of Fusarium oxysporum , F. solani , Pythium ultimum and Phytophthora citrophthora affecting peach seedlings. Results: The in vitro results revealed the ability of these antagonistic in reducing the radial growth of these patho‑ gens. The most important mycelial growth reduction was of 85.82%, recorded for F. oxysporum in confrontation with A5 of Aspergillus candidus . Aspergillus flavus A4 and A. niger A10 were the most effective against F. solani with an inhibi‑ tion percent more than 60%. For P. citrophthora , A. flavus A4, A. candidus A5, A. terreus A9 and A. niger A10 inhibited the mycelia growth by more than 60%. Aspergillus nidulans A1 was the most effective against Pythium ultimum (72.07%). Trichoderma harzianum isolates T9 and T10, are the most effective with a high inhibition percent of mycelial growth. The inhibition induced after 4 days of incubation, against F. oxysporum , F. solani , P. citrophthora and Pythium ultimum by these 2 antagonists exceeded 70, 60, 70 and 80%, respectively. The in planta test showed the efficacy of antago‑ nists tested solo against some pathogens. In fact, Bacillus subtilis improved the health status by 62.55% compared to the control inoculated with P. ultimum . Trichoderma harzianum T9 significantly reduced the root rot index by 87.5% than the control inoculated with F. solani . In the same sense, B. subtilis significantly reduced this parameter by 62.55 and 88.89% than the control inoculated with P. ultimum and P. citrophthora , respectively. Furthermore, B. subtilis (B) and Aspergillus niger A10 improved plants height than the control inoculated with Pythium ultimum by 31.52 and 40.49%, respectively. However, the combinations of antagonists (T9 + T10; A5 + A10 and B + T10) did not improve their efficacy. Conclusions: The isolates T. harzianum (T9 and T10), A. candidus A5 and A. niger A10 were the most effective in vitro against Fusarium , Pythium and Phytophthora species associated with peach seedling decline. The in vivo assay showed the effectiveness of B. subtilis against P. ultimum and P. citrophthora and the potential effect of T. harzianum T9 against F. solani. Their combinations revealed to be ineffective.

Page 2 of 10 Mannai and Boughalleb-M'Hamdi Egyptian Journal of Biological Pest Control (2022) 32:60 et al. 2018a). Complexes of fungi and oomycetes were reported to be the main causal agents of fruit trees decline in orchards and also in nurseries (Mannai et al. 2018a). This complex includes Fusarium spp. Cylindrocarpon spp., Pythium spp., Phytophthora spp. and Rhizoctonia solani (Bent et al. 2009). In Tunisia, previous studies showed that Phytophthora cactorum and many Pythium species such as P. indigoferae, P. irregulare, P. rostratifingens, P. sterilum, P. undulatum were associated with apple trees decline in orchards and showed their virulence (Souli et al. 2014). Prior investigations have demonstrated the association of Fusarium, Pythium and Phytophthora species to apple and peach decline in Tunisian seedling nurseries (Mannai 2019). Chemical products are the most used tools for this disease management. Some fungicides such as fosetyl-Al and metalaxyl showed excellent systemic activity against several diseases caused by Phytophthora and Pythium (Mazzola et al. 2002). In Tunisia, the fosetyl-Al, hymexazol, carbendazim and chinosol were tested in vitro and in vivo and revealed to be effective against Fusarium oxysporum and F. solani associated with the peach seedling decline in nurseries (Mannai et al. 2018b).
However, economic and environmental pressures to reduce the reliance on chemicals have led to a renewed interest in using biological agents such as bacteria and antagonistic fungi. Among biocontrol fungi agents, Trichoderma, Gliocladium and Aspergillus species have been proved to be active to control pathogens in vitro and in vivo (Boughalleb-M'Hamdi et al. 2018). Trichoderma species are the most widely used antagonists for controlling plant diseases (Yassin et al. 2021). Several bacteria such as Bacillus, Pseudomonas, Burkholderia and Streptomyces species have been used to manage soilborne pathogens of many vegetable crops (Mannai et al. 2020). These species are capable of producing some bioactive secondary metabolites (Khan et al. 2020). Several previous studies showed that these fungal and bacterial antagonists' species efficiently controlled Fusarium, Pythium and Phytophthora species pathogens associated with many crops (Mannai et al. 2020). A combinatory approach has also the potential to resolve problems that occur with individual biocontrol agents. Several previous scientists have tested the combination of different biocontrol strains (Thilagavathi et al. 2007).
The objectives of this study were to: (1) evaluate the in vitro antifungal potential of 10 Aspergillus spp. and 10 Trichoderma harzianum isolates against the mycelial growth of F. oxysporum, F. solani, Pythium ultimum and Phytophthora citrophthora isolates associated with peach seedlings decline and (2) study the ability of these antagonists with Bacillus subtilis to suppress and/or reduce the disease severity and enhance the growth of infected peach plants.

Pathogens isolates
The used pathogens for in vitro and in planta assays were isolates of Fusarium and Pythiaceae species obtained from peach tree nurseries in Tunisia. A representative isolate of each species was used (Table 1).

Antagonist's collection
The used antagonistic fungi and the bacterium strain were isolated from the roots and the rhizosphere of apple and peach seedlings from various Tunisian nurseries. The characteristics of all isolates are presented in Table 2.

Evaluation of the effect of the antagonists on mycelial growth of the pathogens associated with peach seedlings decline
Dual culture plate assays were performed in 90 mm Petri dishes containing Potato Dextrose Agar (PDA; Laboratories CONDA; Spain) to study the ability of fungal antagonists to inhibit pathogens mycelial growth. Agar plugs (6  where T is the average colony radius in the presence of the antagonist fungus, and C: average radius of control colonies.

Study of the effect of antagonists on the severity of the peach seedlings decline
The method of Ruano-Rosa and Lopez-Herrera (2009), with minor modifications, was adopted to evaluate the in vivo effect of bioagents on the severity of the peach seedlings decline. For this trial, 2 isolates of Trichoderma spp. (T9 and T10), 2 isolates of Aspergillus spp. (A5 and A10) and B. subtilis strain were used. The inoculum for each oomycete isolate was prepared by growing the pathogen in 500-ml flasks containing 200 g of sand, 20 g of oat and 30 mL of distilled water autoclaved at 120 °C for 20 min on 2 consecutive days. This mixture was inoculated with 10 agar plugs of each isolate per flask under aseptic conditions. To prepare the Fusarium inoculum, bottles containing 200 g of sterile wheat seeds were inoculated with 10 mycelial disks (6 mm diam.) of each Fusarium spp. isolate grown on PDA medium for 2 weeks. Wheat seeds and sand-oat were inoculated with disks of PDA medium served as controls. The flasks were mixed and incubated for one week for oomycetes and 2 weeks for Fusarium spp. at 25 °C and shaken every 2 days to ensure thorough colonization. Sand-oat and wheat seeds inoculums were added to a potting mix (peat and sand in 2:1 v/v) at a rate of 1% (v/v) which was then placed in l0 cm diameter plastic pots. The uninoculated control was an uninfected potting mix.
To prepare the inoculum of each antagonist treatment, a few agar disks from 7-day-old antagonist cultures grown on PDA medium were incubated, for one week, in an Erlenmeyer flask containing 150 ml of Potato Dextrose Broth (PDB; Laboratories CONDA; Spain) medium, with stirring (120 rpm). The suspensions were adjusted to 10 6 spores/ml. Bacillus subtilis cell suspensions used were prepared by scraping bacterial colonies, previously grown in nutrient agar (NA) medium for 48 h, in sterile distilled water (SDW) and adjusted to 10 6 cells/ml. The antagonist treatment was done at the following dates: 1 and 30 days from the start of the experiment (50 ml/ % inhibition = (1 − T /C) * 100 plant). The antagonistic isolates were applied alone and in combinations. The combinations used were: T9 + T10, A5 + A10 and B + T10.
The experiment was conducted according to a completely randomized design, with 3 replicates per elementary treatment. The plants were harvested after 3 months. Four parameters were noted at harvest. These parameters were: the sanitary state index, the seedlings' height, the root weight and the root browning index. The sanitary state index rated onto 0-5 scale (0 = healthy seedlings: 1 = moderate discoloration of plant leaves (≤ 25%); 2 = moderate discoloration and falling leaves (≤ 50%); 3 = moderate discoloration of plant collar, stem and leaves (≤ 75%); 4 = extensive discoloration of plant collar and stem with falling leaves (> %); and 5 = dead plant) (Santini et al. 2006). The root browning index was rated according to a 0-5 scale (0 = no obvious symptoms; 1 = moderate discoloration of root tissue; 2 = moderate discoloration of tissue with some lesion; 3 = extensive discoloration of tissue; 4 = extensive discoloration of tissue with girdling lesions; and 5 = dead plant) (Tewoldemedhin et al. 2011).

Statistical analysis
Data were subjected to a one-way analysis of variance (ANOVA) using Statistical Package for the Social Sciences software (SPSS), version 20.0. The in vitro and in planta tests were analyzed according to a completely randomized factorial model with 2 factors (antagonists and pathogens tested). For all tests, means were compared using Student-Newman-Keuls (SNK) test (P ≤ 0.05).

Effect of antagonists on mycelial growth of the studied pathogens In vitro antifungal activities of Aspergillus species
The mean radius of pathogens colonies, formed after 6 days of incubation at 25 °C, is dependent on antagonistic treatments tested and pathogen isolates, and a highly significant interaction was observed between both fixed factors at P ≤ 0.001. In fact, all Aspergillus species were able to reduce the peach decline pathogens' radial growth compared to their relative untreated controls (Table 3). The most important mycelial growth reduction, compared to the untreated control, was 85.82%, recorded in the case of F. oxysporum treated with A. candidus (A5). The isolates A4 (A. flavus) and A10 (A. niger) were the most effective against F. solani, with an inhibition and A10 (A. niger) inhibited the mycelia growth by more than 60%. The antagonist Aspergillus nidulans (A1) was the most effective against P. ultimum (72.07%) ( Table 3; Fig. 1).

In vitro antifungal activity of Trichoderma harzianum isolates
The in vitro evaluation of the efficiency of different T. harzianum isolates on pathogens hyphal growth showed a highly significant interaction between both fixed factors (antagonists * pathogens) at P ≤ 0.001 (Table 4). The isolates T9 and T10 of T. harzianum, originating from the Kasserine region, exhibited the highest inhibition percent of mycelial growth for different pathogens. The inhibition rates induced after 4 days of incubation against F. oxysporum, F. solani, P. citrophthora and Pythium ultimum by these 2 antagonists were 70, 60, 70 and 80%, respectively (Table 4; Fig. 2). The antagonists A5, A10, T9 and T10 were chosen for the in vivo assays because of their effectiveness against the different tested pathogens.

Effect of antagonists on the severity of peach seedlings decline
The analysis of the relative variance of the health status of the inoculated vegetative part of the plants showed a highly significant difference (P ≤ 0.001) between the different treatments and a significant difference (P ≤ 0.05) between pathogens. Bacillus subtilis (B) improved the health status by 62.55% than the control inoculated with P. ultimum. Other treatments did not significantly improve this parameter for all the studied pathogens (Table 5; Fig. 3). Analysis of the variance relative to the severity of root rot, noted 90 days after planting, showed the existence of a significant interaction (P ≤ 0.05) of the 2 fixed factors (pathogens x antagonists) and a highly significant difference (P ≤ 0.001) between the effect of the tested antagonists. The T. harzianum isolate (T9) and B. subtilis (B) were the most effective. T. harzianum T9 significantly reduced the root rot index by 87.5% than the control inoculated with F. solani. For B. subtilis, this parameter was significantly decreased by 62.55 and 88.89% compared to the control inoculated with P. ultimum and Ph. Citrophthora, respectively (Table 5; Fig. 4). However, no effect was noted on the root rot index when used the other treatments (T. harzianum isolate T10, A. candidus A5 and A. niger A10, A5 + A10, T9 + T10 and B + T10) ( Table 5).
Statistical analysis of the relative variance of plant height showed a significant interaction between fixed factors (pathogens*antagonists) and a highly significant difference (P ≤ 0.001) between different antagonists. The treatments A. niger (A10) and B. subtilis (B) improved the plant height compared to the control inoculated with P. ultimum by 40.49 and 31.52%, respectively (Table 5; Fig. 3). However, all treatments did not improve this parameter for peach seedlings inoculated by F. oxysporum, F. solani or Ph. citrophthora. The seedlings heights were statistically comparable to that of inoculated control ( Table 5).
The analysis of the variance of root weight data revealed a highly significant difference (P ≤ 0.001) according to different treatments and a significant difference between Table 3 Inhibition percentage of peach seedling pathogens colony growth, noted after 6 days of dual culture with Aspergillus spp. isolates *Means ± standard error in the column for each parameter followed by the same lowercase letter were not significantly different according to SNK test at P ≤ 0.01 **Means ± standard error in a row followed by the same capital letter were not significantly different according to SNK test at P ≤ 0.05 ***Probabilities associated with individual F tests   pathogens. However, all treatments did not significantly improve this parameter. They were statistically comparable to the inoculated control (Table 5; Fig. 4).

Discussion
Trichoderma harzianum isolates T9 and T10, native to the Kasserine region, were the most effective on the different pathogens in vitro. The in planta assays demonstrated that T9 significantly reduced the root rot index of peach plants inoculated with F. solani. Several previous studies have shown that the genus Trichoderma is among the most studied biological fungal agents marketed as biopesticides (Jamil et al. 2021 A previous study assessed the potential of four Trichoderma asperellum strains in vitro and in vivo to control Pythium myriotylum, the causal agent of cocoyam root rot disease in Cameroon, showed their efficacy, although differences were found among the strains. In in vivo trials, pretreatment of cocoyam plants with 2 T. asperellum strains reduced P. myriotylum infection by 50% (Mbarga et al. 2012). Moreover, a recent study on the efficacy of T. harzianum and T. viride against Pythium dampingoff of pepper showed that these antagonists inhibited P. ultimum radial growth by 18.54, 17.52 %, respectively, in dual culture assay and reduced the pre-and postemergence pepper damping-off infections (Mannai et al. 2020). Besides, a study established in Saudi Arabia to evaluate the antagonistic efficacy of 2 species of Trichoderma against the most common causative agents of stalk rot disease of maize showed that Trichoderma viride was effective against F. proliferatum and F. verticillioides with mycelial inhibition rates of 80.17 and 70.46% and T. harzianum exhibited rates of 68.38 and 60.64%, respectively (Yassin et al. 2021).
Aspergillus candidus and A. niger were among the most effective isolates to reduce pathogens mycelia growth. In planta, A. niger stimulated the growth of peach plants inoculated with P. ultimum. These results are in agreement with previous studies reporting that several Aspergillus species are capable of producing a number  Page 8 of 10 Mannai and Boughalleb-M'Hamdi Egyptian Journal of Biological Pest Control (2022) 32:60 of bioactive secondary metabolites (Ngo et al. 2021). In addition to their antagonistic capacity, several members of this genus have demonstrated the ability to confer plant resistance to diseases and other known benefits to contribute to general soil suppression (Urja and Meenu 2010).
A previous study proved the efficacy of Aspergillus flavus, A. niger A. terreus and A. fumigatus to reduce the mycelial growth of F. oxysporum and F. solani in vitro and decrease the disease severity and improve the growth of melon and watermelon seedlings inoculated by these pathogens (Boughalleb-M'Hamdi et al. 2018). In the same sense, Dwivedi (2013) confirmed that 4 Aspergillus species (A. flavus, A. niger, A. sulphureus and A. luchuensis) had a fungi toxicity against F. solani. Zhao et al. (2018) proved that a strain of A. tubingensis significantly protected tomato against gray mold, obviously enhanced plant length, dry mass and fresh mass of tomato plants and successfully colonized in the rhizosphere.
The isolate of B. subtilis tested in vivo was also very effective against some pathogens. In fact, this antagonist  Efficacy of B subtilis capacity produces a number of bioactive secondary metabolites. The Bacillus species are the most frequent producers of bioactive microbial metabolites (Saxena et al. 2020). The present results were also in concordance with previous studies showing that the use of B. subtilis, as a soil treatment, might be used to control Pythium and Phytophthora root rot on several crops (Mannai et al. 2020). In addition, B. subtilis cell suspensions decreased the severity of pepper root rot caused by Phytophthora by 55% (Sid Ahmed et al. 2003). In addition, the application of the B. subtilis TM3 formulation was able to suppress F. verticillioides infection in corn plants in Indonesia (Suriani et al. 2021). By contrast, the combinations between antagonists neither reduced the disease severity nor improved the growth of seedlings. Each strain used individually was more efficient than its combination with another antagonist strain. This may be due to an incompatible reaction among strains (Thilagavathi et al. 2007).
Thus, as an eco-friendly alternative to chemical fungicide, the management of the peach seedling decline through biocontrol agents is possible and the results are promising as some of the tested biocontrol agents were effective in reducing disease severity and in improving seedling growth.
Furthermore, in nurseries, seedlings can be attacked by more than one soil-borne pathogen and the disease severity could be due to fungal complexes (Moein et al. 2019). For this reason, it will be important to test these antagonists in nurseries without combinations.

Conclusions
In vitro test of T. harzianum and Aspergillus species proved that the isolates T. harzianum isolates T9 and T10 and A. candidus isolate A5 and A. niger isolate A10 were the most effective against Fusarium, Pythium and Phytophthora species associated with peach seedling decline. The test in planta showed the efficacy of B. subtilis against P. ultimum and P. citrophthora. It reduced the disease severity indexes and improved plants growth. Furthermore, T. harzianum isolate (T9) significantly reduced the root rot index induced by F. solani. Aspergillus niger improved the height of peach seedlings inoculated by P. ultimum. However, their combination uses were ineffective.