Screening and interaction between pathogens and antagonistic seed-borne fungi, associated with some organic spices and vegetable crops in Tunisia

Seed-borne pathogenic and antagonistic fungi were isolated from the same organic vegetable and spice seeds (carrot, fennel, broad bean, faba bean, and lettuce), and the interaction between them were studied as a part of a biocontrol assay. In vit ro dual culture assay between each pathogen and the antagonists on PDA medium was made, which led to the inhibition of the pathogens mycelial growth. According to results generated for fennel seed-borne fungi, direct confrontation of the five pathogens ( Botrytis cinerea , Sclerotinia sclerotiorum , Cladosporium cladosporiodes , Cladosporium link and Bispora sp.), with Aspergillus niger and Penicillium digitatum showed an inhibition of radial growth above 50%. For lettuce; the highest inhibition was recorded by Trichoderma harzianum in confrontation with S. sclerotiorum , and P. digitatum in confrontation with Alternaria alternata . For carrot, three fungal pathogens were identified as: A. alternata , A. solani and Pythium sp., which were faced with three antagonists ( P. chrysogenum , A. niger and T. harzianum ), and showed radial inhibition about 50%. Concerning broad bean and baba bean; only A. alternta was isolated, and has been confronted with T. harzianum , P. digitatum , and P. italicum . During in vivo assays on fennel plant in the greenhouse; a disease severity index caused by various pathogens alone and in combination with antagonists compared to control plants, was recorded. According to results observed, fennel plants inoculated with B. cinerea and treated curatively with P. digitatum and preventively with A. niger ; recorded a low disease severity index value of 0.22. This work was carried out to find effective seed-borne bioagents that could cause in vitro and in vivo inhibition of the growth of several fungal pathogens of vegetables; thus could be applied in the field for the biocontrol of fungal spice and vegetable crops diseases.


Introduction
Fungi constitute the largest group among seedborne pathogens. Many fungal pathogens infect the developing and maturing seeds, and reduce their yield both quantitatively and qualitatively. Furthermore, seed-borne fungi can be transmitted from the seed to plants through infecting the growing seedlings (Neergaard, 1977). Early detection of seed-borne fungal pathogens was particularly important, as infected seeds often appear symptomless. Thus, diagnosis of infected seeds can avoid uncontrolled propagation of pathogens throughout long distances when exchanging such seeds (Manici et al., 2016).
Seeds represent a particularly efficient vehicle to disperse seed-borne pathogens. A seed-borne pathogen growing externally, internally or associated with the seed as contaminant might cause; seed abortion, rot, necrosis, reduction or elimination of its germination capacity. As a result, seedling damage causes development of plant disease at later stages of its growth (Khanzada et al., 2002). The most predominant fungal genera encountered on spice seeds were Aspergillus sp., Penicillium sp., Alternaria sp., and Fusarium sp. Detection approaches of the fungal seedborne pathogens have been developed using different technologies. Manici et al., (2016) reported that accurate and rapid identification of these pathogens was essential to satisfy phytosanitary regulations, and consequently control diseases effectively. The use of alternative control methods such as biological control for suppressing seed-borne fungi have been developed rapidly, and were playing an important role in reducing the use of pesticides. Most of the studies on biocontrol of seed-borne fungal diseases have focused on several cereal crops such as; wheat, barley, rice, sorghum, cowpea, and soybean, as well as on muskmelon, cabbage, carrot and onion within the vegetable crops (Özer and Coşkuntuna, 2016). According to Özer and Coşkuntuna, (2016), successful implementation of biological control using different bioagents were promising for seed-borne fungi, because of the hazards of chemical control for the growers, consumers and the environment as well. Seeds treatment with beneficial microorganisms including fungi and bacteria (i.e. Trichoderma, Pseudomonas, Bacillus, Rhizobia, etc.), could improve a wide variety of biotic, abiotic, and physiological stresses to the seeds and seedlings (Mastouri et al., 2010). Moreover, these bio-agents were capable of enhancing, promoting seed germination and subsequent plants growth (Moeinzadeh et al., 2010). Trichoderma spp. were common in all soils and root ecosystems, have been recognized to control several soil-borne fungal genera of Fusarium, Pythium, Sclerotinia, Rhizoctonia, Gaeumannomyces and others (Howell, 2003). The biocontrol efficacy of these microorganisms last longer than that of synthetic chemicals, and can therefore protect the plant throughout all its growth stages (Rojo et al., 2007).
The aims of the current work were to; i)-isolate and identify seed-borne fungi from organic spices and vegetables, ii) study the in vitro and in vivo interaction between antagonists and certain fungal pathogens to detect their antifungal potentialities, thus could be used for biocontrol of several fungal diseases affecting these crops in the field.

Screening of seed-borne fungi of spice seeds
Organic seeds samples of carrot, fennel, broad bean, faba bean, lettuce and coriander were provided by the technical center of organic agriculture (CTAB, Tunisia). The collected seeds were analyzed for the presence of seed-borne fungi. Two hundred seeds were tested for each variety with four replications. The seedborne fungi were isolated using two methods according to Walcott, (2003) with some modifications: Novel Research in Microbiology Journal, 2019 1)-Twenty-five seeds of each plant were placed on three layers of sterile moist blotting paper (ISTA, 1996) in each glass Petri dish. These Petri dishes were incubated at 25±1°C under 12/12 h light and darkness cycle for 7 days. The germination rate (GR) and incidence rate (IR) were calculated for each sample according to these two formulas; GR = (Number of germinated seeds/ Total number of seeds examined) × 100, whereas, IR = (Number of infected seeds/ Total number of seeds examined) × 100. Each seed was observed under stereomicroscope to detect the presence of fungal growth.
2)-The second method was agar plate method. Surface disinfected seeds (placed in 0.1% mercuric chloride for 30 sec., then washed three times in sterile dist. water) were plated on the surface PDA medium, and then incubated for 5-7 days at 22-25°C under 12h altering cycles of light and darkness. After incubation, fungi growing out from the seeds on the agar medium were examined, identified and monitored. The percentages of isolated mold fungi were expressed as isolation rate %, ISR = Count of detected fungal colonies appeared on each seed/ Total count of colonies × 100. Fungal identification was carried out based on colonies and microscopical characteristics (Hedawoo et al., 2014).

Detection of antifungal activities
The antagonists isolates identified were tested for their in vitro and in vivo antifungal potential against the seed-borne fungal pathogens recovered from the same seeds.

In vitro dual culture assay
Mycelial disc (4 mm diameter) obtained using sterile cork borer from the growing edge of five days old of each antagonist's culture; was placed in front of a pathogen disc at the opposite side of the PDA plate, 3 mm from the periphery. For control, mycelial plug of a pathogen colony was placed singly at the center of a PDA plate according to Boughalleb-M'Hamdi et al., (2018). Three replicates were used for each treatment (pathogen/ antagonist). Plates were incubated at 28ºC for 5-7 days, and then the diameter of radial mycelial growth of each pathogen was measured. The inhibition percent was determined referring to the formula of Hmouni et al., (1996): IP (%) = (1-C n /C 0 ) x 100; where C n is the radial growth of the pathogen in the presence of the antagonist and C 0 is the radial growth of the pathogen without antagonist.

In vivo antifungal potency
This experiment was carried out in the greenhouse and concerned only with fennel plants. Pre-germinated seeds of fennel were sowed in indented trays containing sterilized peat. Growing seedlings were placed in a pot containing a mixture of peat and vermiculite (1:1); at the rate of 3-5 seedlings in each pot. The inocula of pathogenic Botrytis cinerea and Sclerotinia sclerotiorum, and those of the fungal antagonists (P. digitatum and A. niger) were prepared as follows: Fungal cultures were grown on PDA at 28°C for 5 days, and then Erlenmeyer flasks containing 50 ml of potato dextrose broth (PDB, 20 g/l) were inoculated separately with four discs (1 mm) of each isolate. Flasks were incubated at 28°C for 5 days in an orbital shaker, and then conidial suspensions were recovered from each broth culture by filtration. Conidial suspension of pathogens were adjusted to 10 6 cfu/ml, whereas, those of antagonists were adjusted to 10 8 cfu/ml using haemocytometer. Application of each fungal antagonist was carried out separately 24 h before artificial inoculation of each pathogen as preventive treatment, and 24 h after inoculation of each pathogen as curative treatment, with the two antagonists applied separately and on combination. The assay was carried out by spraying fennel leaves of each seedling by the same amount (10 ml) of each fungal antagonist and pathogen suspension, separately. The Novel Research in Microbiology Journal, 2019 pots were then placed in a greenhouse for 45 days. Controls were performed by spraying the plant with pathogen only in case of positive control, and with dist. water as negative control. For each treatment of pathogen and fungal antagonist; fennel plants were randomly distributed with 5 plants per replicate (3 replicates). Disease index was measured within 6 weeks following inoculation. Areas of observed symptoms were scored for disease index using a scale from 0 to 4; 0 = no symptoms; 1 = few scattered lesions covering about 1-10% of the leaf; 2 = spots covering about 11-25% of the leaf; 3 = spots coalescing and covering about 26-50% of the leaf according to Mostafa et al., (2013).

Statistical analysis
The data were analyzed by ANOVA using SPSS version 20.0 statistical software (SPSS, SAS Institute, USA), to evaluate differences between parameters. Differences between treatments were determined by Duncan multiple range test at 5% of significance level.

Screening of seed-borne fungi
Isolation percentage and identification of fungal species recovered from vegetables and spices seeds were presented in Table (1). Seed samples were analyzed for incidence rate, isolation rate, and germination rate ( Table 2). Percentage of seed infection recorded were in the range between 17.14 (faba bean) and 68.57% (coriander). All seven samples were infected with fungi. The highly contaminated seeds were observed in fennel (87.55%) and carrot (85.71); followed by lettuce of 82.22% then broad bean with 77.43%. While the lowest contaminated seeds were in those of faba bean with 50%. The seed germination ability was significantly reduced by all fungi, and recorded data were between 33.33 (fennel) and 65.71% (faba bean) (p<0.05). A total of 16 fungal species were recovered and identified (Table 3). The highest isolation percent of pathogens was recorded for A. solani (40%) from carrot, and A. alternata from faba bean. Other predominant pathogens recovered on fennel were S. sclerotiorum (33%) and (48%) on lettuce, Botrytis cinerea (28%) on fennel. T. harzianum was also detected from carrot and lettuce seeds with values of 30, and 20%, respectively. A. niger was isolated from carrot (40%), fennel (33%) and lettuce (48%) seeds.

In vitro antifungal potential
Statistical analysis revealed that fungal bioagents inhibited the mycelial growth of all pathogens with different values as presented in Table (3). Growth inhibition of A. alternata isolated from carrot, lettuce, broad bean and faba bean seeds differed significantly (p < 0.05). T. harzianum and P. digitatum displayed a potent antifungal activity against A. alternate, with values varying from 72.31 (carrot) to 81.22 (lettuce) for T. harzianum, and from 79.37% (broad bean and faba bean) to 84.53% (lettuce) for P. digitatum. These results demonstrated that both were most potent bioagents. S. scletiorum isolated from lettuce seeds (p < 0.05) exhibited a moderate resistance against A. terreus (36%), and A. flavus (37%). P. digitatum and A. niger reduced the in vitro radial growth of B. cinerea, S. sclerotiorum, C. cladosporioides, C. link and Bispora sp. isolated from fennel seeds with varying degrees (Table 3).

In vivo antifungal potency
The effectiveness of inoculation of fennel plants with different fungal antagonists were estimated in vivo using the disease severity index data. Results recorded a significant difference between treated plants by the two pathogens (B. cinerea and S. sclerotiorum), and the two antagonists (P. digitatum

Discussion
Screening of the main seed-borne fungi is an important tool in the seed health research. In the current work, some pathogenic genera with important frequencies were isolated and identified including; Botrytis cinerea, Sclerotinia sclerotiorum, Cladosporium cladosporiodes, Cladosporium link and Bispora sp. were predominant. Seeds screening showed prevalence of A. solani and T. harzianum from carrot seeds; A. alternata and P. italicum from faba bean and broad bean seeds, S. sclerotiorum and T. harzianum from lettuce seeds, Alternaria alternata, Fusarium oxysporum f.sp. funiculi; and other genera like Aspergillus, Penicillium, Cladosporium, Fusarium from fennel seeds. Similar results were obtained previously by Odstcilova et al. (2002) who observed the presence of 26 fungal species associated with fennel seeds in Czech Rebublic. Verticillium dahliae was the most frequent seed-borne fungus recorded in the study of Ghoneem et al., (2009). More recently, Hedawoo et al., (2014) have isolated 12 fungal species associated with seeds of black pepper; 11 isolates on fennel, while eight on cumin. A. flavus, A. fumigatus, A. niger, Rhizopus nigricans and C. cladosporioides were the predominant fungi on all of the three tested seeds. Prevalence of mycoflora of black pepper, fennel and cumin seeds had been highlighted also by Hedawoo and Chakranarayan, (2011); Ramesh and Jayagoudar, (2013) . Pant, (2011) reported the occurrence of species of Alternaria, Aspergillus, Curvularia, Rhizopus and Mucor on coriander seeds.
Biocontrol agents have been reported as suitable for controlling seed-borne fungal diseases. In nature, antagonists exist in association with different microbes in various forms such as; antibiosis, symbiosis and synergism (Baig et al., 2012). In this present study, seven antagonists were confronted with the main seed-borne pathogens isolated from organic seeds to prove their antagonistic potentiality. Tapwal et al., (2015)   inhibitory actions on the growth of the selected seed borne pathogens to a varied extent. Similarly in our work; selected species of Trichoderma, Aspergillus or Penicillium showed different levels of inhibitory potential against five selected seed borne pathogens. Inhibition of plant pathogenic fungi by different species of Trichoderma was studied under in vitro conditions by several researchers (Boughalleb et al., 2008;Shaikh and Sahera, 2013;Enespa and Dwivedi, 2014 Currently, the in vivo biocontrol potency of P. digitatum and A. niger against B. cinerea and S. sclerotiorum isolated from fennel seeds was recorded. Both antagonists proved to be most effective preventively and curatively when treated alone, or in combination showing a lower disease severity index. Earlier, Sutton and Peng, (1993) revealed the efficacy of Penicillium sp. against B. cinerea suppressing the sporulation incidence of this pathogen by 64%. The mycoparasitic potential of Aspergillus species against several aerial and soil borne plant pathogens led to the development of biocontrol strategies by this antagonist (Dubey et al., 2007;Hajieghrari et al., 2008). However, although Aspergillus spp. and Penicillium spp. were effective against the tested phytopathogens, these fungi were not recommended for in vivo biocontrol assays due to their carcinogenic properties (Boughalleb-M'Hamdi et al., 2018).
Novel Research in Microbiology Journal, 2019

Conclusion
The improper and poor storage conditions, as well as traditional agricultural practices resulted in contamination of seeds, thus become unfit for germination. In the present study, the seed health status was assessed by studying the potential of several fungal bioagents to antagonise the major seed-borne pathogens of certain spice and vegetables. These bioagents proved their efficiency to reduce the in vitro, and in vivo mycelial growth of the fungal pathogens. Growers are thus advised to use these bioagents to treat their seeds before sowing in the field, as such approach will reduce disease incidence. This work presented the first screening of seed-borne fungi from local spices and vegetable seeds in Tunisia.