Application of different combinations of lactic acid, phototrophic bacteria and yeast mixtures in control of seed and seedlings pathogens of tomato and pepper

Application of three combinations of lactic acid bacteria (Lactobacillus
 plantarum, Lactobacillus rhamnosus), phototrophic bacteria (Rhodopseudomonas
 palustris) and yeast (Saccharomyces cerevisiae) with sugar cane molasses,
 marked as: EM1, EM5 and EM AGRO, against the phytopathogenic fungi of tomato
 and pepper: Fusarium oxysporum, Alternaria alternata, Botrytis cinerea,
 Colletotrichum sp., Verticilium dahliae and Pythium aphanidermatum was
 evaluated in vitro and in vivo. A combination of bacteria and yeast named
 EM5 showed the highest mycelium growth inhibition against B. cinerea (38.4%)
 in a double agar diffusion test. In a microdilution test, the combination
 EM1 showed the highest inhibitory effect on B. cinerea (MIC 1x10-3 ?l/ml),
 while EM5 showed a similar inhibitory effect towards F. oxysporum, A.
 alternata and Colletotrichum sp. (MIC 10 ?l/ml). The use of EM1 (in
 concentrations 10 and 100 ?l/ml) and EM AGRO (10 ?l/ml) is recommended for
 tomato seedling protection. ??1 (100 ?l/ml), ??5 and ?? AGRO (10 ?l/ml) are
 recommended for pepper seedling protection.


INTRODUCTION
Tomato (Solanum lycopersicum L.) and pepper (Capsicum annuum L.) are two very important vegetable crops in Serbia. In 2019, tomato production in Serbia was 111.649 tonnes on 7.880 ha (FAO, 2021), while Gvozdenović (2010) reported over 150.000 tonnes of peppers that were harvested from 21.000 ha.
Tomato and pepper crops are exposed to many phytopatogenic fungi, such as: Alternaria alternata, Colletotrichum spp., Fusarium spp. (Mannai et al., 2018;Rezaee et al., 2018), Pythium spp. (Whipps & Lumsden, 1991), Botrytis spp. (Williamson et al., 2007), Rhizoctonia spp., Septoria lycopersici and Verticilium spp., which are able to cause severe economic losses. Some of these phytopatogenic fungi can produce toxins that have harmful consequences for human health. Frequent application of synthetic pesticides, as control measures in the management of seed and seedlings diseases, is associated with resistance of these pathogens to synthetic pesticides (Rosslenbroich & Stuebler, 2000;Hahn, 2014), which increases production costs and polluting the environment.
Biological control is one of the most important alternative strategies (Karimi et al., 2012). The issues of fungal resistance, environmental pollution, and negative effects on human health can be significantly reduced by applying biological plant protection products. Several bacterial antagonists are used in plant protection, but as they live in nature close to pathogens, they need to be identified, isolated, amplified and correctly applied.
Important groups of microorganisms used in the biological control of fungal diseases are lactic acid bacteria (LAB) (Dalie et al., 2010;Laref & Guessas, 2013;Zebboudj et al. 2014). The application of plant growth promoting bacteria (PGPB), to control phytopathogens, has gained increasing attention, for example purple nonsulfur bacteria (PNSB) Rhodopseudomonas palustris strains have been mentioned as possible biocontrol agents (Nookongbut et al., 2019). Therefore they may be considered as commercial alternatives to chemical pesticides to manage plant diseases, provide food security and contribute to a sustainable agrosystem (Stamenković et al., 2018).
The objective of this study was to determine the antagonistic capacity of PGPB by evaluating the antifungal power of three combinations of lactic acid bacteria, a phototrophic bacterium and yeast in vitro and in vivo against the phytopathogenic fungi of tomato and pepper: Fusarium oxysporum, Alternaria alternata, Botrytis cinerea, Colletotrichum sp., Verticilium dahlia and Pythium aphanidermatum.

Antagonistic activity of investigated mixtures
Double agar diffusion test. To evaluate the efficiency of three combinations of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus rhamnosus >10 3 CFU/g), phototrophic bacteria (Rhodopseudomonas palustris >10 3 CFU/g) and yeast sugar molasses (Saccharomyces cerevisiae >10 3 CFU/g), marked as: EM1, EM5 and EM AGRO (property of LUMAX -doo, Belgrade, products registered commercially as soil conditioners ), an in vitro assay was performed on potato dextrose agar (PDA) to observe mycelial development of F. oxysporum, A. alternata, B. cinerea, Colletotrichum sp. (from a collection of the Institute for Plant Protection and the Environment, Belgrade), V. dahliae, and P. aphanidermatum (from a collection of the Institute of Pesticides and Environmental Protection, Belgrade), originating from tomato and pepper seeds. Mycelial disks (5 mm diameter) from 15-day old pure cultures of the investigated fungi were placed on PDA dishes. Twenty-four hours later, different bacterial combinations were added at 3 cm distance. Petri dishes with mycelia disks alone served as the positive control (K+). The dishes were incubated at 25°C. Three replicates were used for each treatment. After 7 days, mycelia diameter was measured in two directions. The percentage of inhibition of radial growth (PIRG) was calculated following the method of Al-Al-Hetar et al. (2011): where R1= radial micelial growth on the control plate, and R2 = radial micelial growth on treated plates.
Microdilution test in vitro. Minimum inhibitory concentrations (MIC) of three combinations of lactic acid, phototrophic bacteria and yeast, marked as: EM1, EM5 and EM AGRO against F. oxysporum, A. alternata, B. cinerea, Colletotrichum sp., V. dahliae,. and P. aphanidermatum, were determined by microdilution using 96-well microtitre plates according to Balouiri et al. (2016) in a concentration range of 10 μl/ml -1×10 -9 μl/ml of each mixture. Fungal spores were washed from the surface of potato dextrose (PD) plates with sterile 0.85% saline solution containing 0.1% Tween 80 (v/v). Spore suspension was adjusted to a concentration of approximately 5×10 4 in the final volume of 100 μl per well with different dilutions of bacterial suspension. Microtitar plates were incubated for 5 days at 25°C. The experiment was repeated four times. Fluconazole (0.8 mg/ml) was used as a positive control. The lowest concentrations without visible growth were defined as the minimum concentrations inhibiting fungal growth.

Effects of tested mixtures on seed and seedling infection percentage
Filter paper test. Effects of two concentrations (100 μl/ ml and 10 μl/ml) on the percentage of infection of tomato and pepper seed on filter paper were examined. Sixty seeds (20 in each of three repetitions) were soaked in the two concentrations and transferred to wet filter paper for two exposure periods lasting 3 h and 4 h. The percentage of infection was assessed 7 days after treatment. Seeds soaked in sterile water were used as a negative control.
In vivo (soil test). Untreated seedlings of tomato and pepper were planted in soil substrate, watered with 3 ml of tested mixtures at concentrations of 100 μl/ml and 10 μl/ ml every 4 days during three weeks. The experiment was set up in three replications with 20 plants in each variant. An untreated control was watered with the same amount of water. The presence of disease was recorded after 15 days. The results were analysed using the statistical analysis package STATISTICA c. 6 (StatSoft, Inc.).
This experiment demonstrated the highest susceptibility of V. dahlia and B. cinerea (<1 μl/ml) to all tested mixtures, while F. oxysporum and P. аphanidermatum showed   (Figures 2 and 3).

Influence of tested combinations on infection percentage of seeds and seedlings of tomato and pepper
Effects of tested combinations on the percentage of infected tomato and pepper seeds (on filter paper). Experiment analysis showed that 15 of 20 tomato plants in the non-treated experiment were asymptomatic on average, while 19-20 of 20 plants (per repetition) were asymptomatic in the treated plates (Figure 4).
An analysis based on concentration and exposure time of seedlings to combinations revealed that an average of 15 pepper seedlings were asymptomatic in the non-treated control, while the number ranged from 15-20 (20 seedlings per repetition) in treatments. Only seedlings treated with EM AGRO at 100μl/ml concentration were infected as high as control seedlings, while all other treatments showed a significant decrease in infection ( Figure 5).

Effects of tested combinations on the percentage of infection of tomato and pepper seedlings (soil test).
The EM1 and EM5 treatments applied at both concentrations completely suppressed the occurrence of Fusarium sp. The treatment with EM AGRO completely suppressed the occurrence of Pythium sp. in tomato seedlings ( Figure  6A). The most effective treatment was EM1 at both concentrations as it managed to suppress the occurrence of fungi of the genus Fusarium, as well as fungi of the genus Pythium, which appeared in 5% of the samples, while 20% appeared in control samples). Data analysis ( Figure 6B) showed a statistically significant increase in the number of asymptomatic plants (14-18) treated with any of the three combinations, while an average of 9 asymptomatic plants were observed in the non-treated control.
The infection rate of Fusarium in non-treated control was 85%, while treatments with EM1 and EM5 at 100 μl/ml concentration completely suppressed these phytopatogenic fungi in pepper seedlings ( Figure 7A). The highest efficacy in suppressing fungi of the genus Pythium was observed in the treatments EM1 and EM AGRO at 10 μl/ml concentration. Data analysis showed that pepper seedlings treated with any of the three combinations showed statistically significant 17-18 asymptomatic plants of the 20 tested, while an average of 4 asymptomatic plants were observed in the control treatment ( Figure 7B).

DISCUSSION
New, alternative strategies for biological control using lactic acid bacteria have been explored to understand the relation between pathogens and antagonistic bacteria in order to control many phytopatogenic casual agents, for example: Fusarium spp. (Lavermicocca et al., 2000), A. alternata (Zabouri et al., 2021), B. cinerea, Monilinia laxa, and Penicillium expansum (Trias et al., 2008).
Elsewere, the application of R. palustris as a plant growth promoting bacteria (PGPB), was shown to influence plant growth and combat plant patogens, such as Magnaporthe oryzae (Nookongbut et al., 2020). Nally et al. (2012) published important data about the antifungal activity of yeast, S. cerevisiae, against B. cinerea on grapes, while Chand-Goyal and Spotts (1997) and Spadaro et al. (2004) examined it on apples not only at room temperature, but also in a refrigerated chamber. Several reports have mentioned the potential use and applications of different genera and species of antagonist yeasts to control B. cinerea on grape tissues (Lima et al., 1999;Castoria et al., 2001;Zahavi et al., 2000;Schena et al., 2000;Masih et al., 2001;Sesan et al., 1999). Other researchers have also reported biocontrol potentials of S. cerevisiae against Penicillium roqueforti in stored wheat (Petersson & Schnurer, 1995), Macrophomina phaseolina and Fusarium solani in tomato (Attyia & Youssry, 2001),  Monilia fructicola in apples (Zhou et al., 2008) and A. alternata in Pinus silvestris (Payne et al., 2000). There are no reports in literature about combined antagonistic effects of lactic acid, phototrophic bacteria and yeast. The results of this study showed that a combination of different lactic acid bacteria (L. plantarum, L. rhamnosus), phototrophic bacteria (R. palustris) and yeast (S. cerevisiae), marked as EM5, demonstrated a strong antifungal effect against F. oxysporum, A. alternata and B. cinerea. The combination EM5 showed the highest rate of spore inhibition towards F. oxysporum, A. alternata, B. cinerea, Colletotrichum sp. and P. aphanidermatum. All three combinations, at both concentrations and exposure times, showed significant decrease in infection of tomato seeds on filter paper. The treatment EM1 applied at 10 µl/ml concentration over 3 h exposure time, and EM AGRO concentration of 10 µl/ml and 4 h exposure time achieved complete symptom suppression on pepper seeds on filter paper. Both concentrations of all three tested combinations reduced the percentage of tomato and pepper infection with the phytopatogenic fungi Fusarium sp. and Pythium sp.
Both concentrations of EM1 treatment showed significant efficacy on tomato, and 100 µl/ml concentration on pepper, as well as the lower concentration (10 µl/ml) of EM AGRO on tomato, and the lower concentration (10 µl/ml) of EM5 and EM AGRO on pepper.
Determination of efficacy of biological agents is of paramount importance for preserving ecosystem and human health, and represents the first step towards implementation of alternative, non-pesticide methods in plant protection.
A combination of bacteria and yeast named EM5 stood out in our current in vitro experiments as the combination with the highest antifungal potential.
In situ experiments on tomato and pepper seedlings showed a high potential of all combinations used, especially the lower concetrations (10 µl/ml), while the lowest rate of seedlings infection was achieved by applying the combination of EM1 (10 µl/ml-3 h) and ЕМ AGRO (10 µl/ml-4 h).
The use of EM1 (at both concentrations) and EM AGRO (10 µl/ml) is recommended for tomato seedling protection. ЕМ1 (100 µl/ml), ЕМ5 and ЕМ AGRO are recommended to be used at lower concentration (10 µl/ ml) for pepper seedling protection.
The results obtained from in situ and in vitro experiments represent the basic principles for synthesizing biological plant protection products based on the tested combinations of bacteria and yeast, which could safely reduce the infection potential of important phytopathogenic fungi: F. oxysporum, A. alternata, B. cinereа, Colletotrichum sp. and P. aphanidermatum. Primena različitih kombinacija smeša mlečno kiselinskih, fototrofnih bakterija i kvasaca u suzbijanju patogena semena i klijanaca paradajza i paprike