In vitro and in vivo effects of Pseudomonas spp. and Bacillus sp. on Fusarium acuminatum, Botrytis cinerea and Aspergillus niger infecting cucumber

Jasmina Zdravković1*, Milan Ugrinović1, Milan Zdravković1, Slaviša Đorđević2, Snežana Pavlović and Dragana Jošić3 1 Institute of Vegetables, Karadjordjeva 71, 11420 Smederevska Palanka, Serbia 2 University of Belgrade, Faculty of Agriculture, Nemanjina 8, 11080 Zemun, Serbia 3 Institute of Soil Research, Teodora Drajzera 7, Belgrade, Serbia (*jzdravkovic@institut-palanka.co.rs) Received: April 15, 2015 Accepted: June 19, 2015


IntroductIon
include bacterial species that live in close association with plants and have beneficial effects on their host. PGPR affect plants in various ways: they synthesize the compounds necessary for plant nutrition, facilitate the absorption of certain nutritional substances from soil, and reduce or prevent the sensitivity of plants to soil pathogens, inducing their systemic resistance. The influence of PGPR can be direct (promoting plant growth through plant hormone production and absorption of nutritional substances) and indirect (prevention of harmful effects of pathogenic microorganisms) (Hayat et al., 2010). Biological products for vegetable nutrition based on PGP microorganisms are considered to be an alternative to chemical fertilizers. Microbiological treatment can stimulate plant growth and considerably decrease the amount of fertilizers necessary in growing substrates (Sripontan et al., 2014), both in organic and conventional systems of vegetable growth. Beneficial microorganisms, such as biological control agents (BCAs) and PGPRs, can play a key role in this major challenge (Raaijmakers et al., 2009). Strains of to the genera Bacillus and Pseudomonas have been used in experimental tests on a wide range of economically important crops (Kokalis-Burelle et al., 2006;Meyer et al., 2010). The PGPR included indigenous strains belonging to Pseudomonas and Bacillus genera selected from different soils in Serbia. Those strains have exhibited numerous PGP traits (Djuric et al., 2011;Josic et al., 2012), stimulated plant growth (Jarak et al., 2012), and some of them demonstrated an ability to inhibit several phytopathogenic fungi (Jošić, et al., 2012 a,b,c;Protolipac et al., 2012;Starovic et al., 2013).
Cucumber (Cucumis sativus L) is one of the most popular members of the family Cucurbitaceae and the second most important vegetable crop after tomato in Western Europe (Eifediyi & Remison, 2010). Historical records about cucumber cultivation date back 5000 years (Wehner & Guner, 2004). Many pathogens of this plant affect its growth and yield. Fusarium spp. are among the phytopathogenic fungi that cause deleterious effects on cucumber plants and yields. These fungi survive and overwinter in greenhouse and field soils for a long time and it is very difficult to eliminate them from soil by chemical fungicides. Cultural practices and biological control measures have been reported as successful in minimizing such deleterious effects of these fungal pathogens (Ling et al., 2010;Eifediyi & Remison, 2010;Cao et al., 2011). Fusarium acuminatum Ellis & Everhart causes diseases of many plants, including those in the Cucurbitaceae family. This is a toxic fungus than can be infectious both for people and warm-blooded animals. F. acuminatum produces the T-2 toxin, which is the most toxic representative among trichothecene mycotoxins (Wing et al., 1994). Grey mould disease caused by the necrotrophic fungus Botrytis cinerea Pers is a devastating agent of more than 200 plants, including vegetables. Grey mould is economically very significant since it causes great losses, especially in vegetable and fruit production. B. cinerea is controlled mainly by applying fungicides, but biological control has recently become a sound alternative to chemicals (Couderchet, 2003). The ochratoxin A-producing Aspergillus niger has also been isolated from cucumber fruits. A. niger was found as a saprophyte in the seed of many field crops, vegetables and herbs (Stević et al., 2012). It causes grey mould in fruits and vegetables during their shelf life and transport. A. niger produces several micotoxins, including ochratoxin A as the most important one (Vuković et al., 2009).
The aim of this study was to investigate the effects of several bacterial strains on growth inhibition of phytopathogenic fungi isolated from cucumber: F. acuminatum, B. cinerea and A. niger, as well as their promoting effects on the cucumber plants. Bacterial antagonists that belong to Pseudomonas and Bacillus genera were used as a mixture for plant growth promotion and protection. We tested their effects on growth dynamics of transplanted plants, on the nitrogen balance index (NBI) and the content of chlorophyll in transplanted cucumber plants. The ultimate objective was to find optimal combinations of bacteria for formulating a microbiological fertilizer for cucumber seedling production.

Bacterial strains and their antagonistic activity to phytopathogenic fungi isolated from cucumber
The bacterial strains Bacillus sp. Q10, Pseudomonas chlororaphis Q16, and Pseudomonas sp. K24, K27, K35 and N3 were isolated from different soils in Serbia. Taxonomic characterization of the isolates was performed based on tests for Gram staining, cytochrome oxidase, catalase, fermentation/utilization of glucose, lactose and sucrose, utilization of citrate, ability to degrade urea and 16S rDNA analyses (unpublished data). The bacteria were grown in liquid King's B medium (KB) for 24 h on orbital shaker at 120 rpm. Concentrations were determined spectrophotometrically (OD 600 ) and all experiments were conducted using 10 6 CFUml -1 .
The phytopathogenic fungi F. acuminatum, B. cinerea and A. niger were isolated from cucumber plants and fruits (private collection of S. Pavlović). The antagonistic activity was tested on Waksman agar (WA) (Wolf et al, 2002) using overnight cultures of bacteria optimized to 10 6 CFUml -1 . The influence of: a) the whole culture (PK) b) extracellular metabolites (supernatant) (SN) and c) heat stable antifungal factors (HSAFs) were tested on the phytopathogenic fungi. SN was obtained by centrifugation of overnight bacterial cultures at 10000 rpm for 8 min. HSAFs, as the termostable extracellular metabolites, were obtained after heat treatment of bacterial supernatant at 70 o C for 30 min. Bacterial agents (10µl) were applied near the edges of Petri dishes, while 6 mm plugs of fungal mycelia were placed at the centre. Negative control variants contained only mycelia on WA plates. After seven days of incubation at 26°C, fungal growth was measured and the percentage of growth inhibition was calculated using the formula: % Inhibition = [(R-r)/R] x 100, where r is the length of the fungal colony opposite the bacterial colony, and R is the maximum radius of fungal colony in the control (Zarrin et al., 2009).

Plant material and pot experiments
The effects of PGP rhizobacteria were tested on cucumber (Cucumis sativus cv Renesansa F1), in a glasshouse of the Institute for Vegetable Crops, Smederevska Palanka, Serbia. Four experimental variants were set up in ten replications: 1. K -Control; 2. M3mixed Bacillus sp. Q10 and P. chlororaphis Q16; 3. M4mixed Bacillus sp. Q10 and Pseudomonas sp. K24; and 4. M5 -mixed Bacillus sp. Q10 and Pseudomonas sp. K35. The mixture of Bacillus sp. Q10 and Pseudomonas sp. N3 (M1), as well as the mixture of Bacillus sp. Q10 and Pseudomonas sp. K27 (M2) were excluded from further experiments because of their low antagonistic activities against the tested fungi in vitro. The seedlings were transplanted on April 9 th to pots sized 10x10 cm, containing the commercial substrate Gramoflor GmbH&Co.KG, Germany, (250 l). Treatment with 3 bacterial mixtures (M3, M4 and M5) was performed 2 days and another treatment one month after sowing. Negative control was treated with the same volume of distilled water. Plant height as a parameter of cucumber growth dynamics was measured on 5 dates following the second treatment (on May 8 th , May 12 th , May 18 th , May 25 th and June 1 st ). Seedlings were grown according to standard growing methods until June 1 st when all other relevant parameters were measured as well, namely: • Plant height, root length, root mass, number of formed leaves, plant weight, leaf weight; • Nitrogen balance index (NBI) and chlorophyll level by polyphenol and chlorophyll meter Dualex scientific 4+, Force A, France.

data analysis
Plant growth dynamics (plant height) after the second treatment was followed on a trend line. The line represents the average state of the observed phenomena trough time. The following model of linear trend (Njegić et al., 1991) was applied: Differences among the treatments (K, M3, M4 and M5) regarding the several morphological traits were shown by mono-factorial ANOVA, while the least significant difference (LSD) test was applied to determine the least significant difference between the treatments and control. LSD was calculated at p ≤ 0.05 according to Gomez and Gomez (1984).

reSultS antagonistic activity of Pseudomonas spp. to phytopathogenic fungi isolated from cucumber
The inhibitory activity of Bacillus sp. Q10 and five Pseudomonas spp. against fungal pathogens responsible for detrimental effects on cucumber plants and fruits were assessed using the dual culture method. Three phytopathogenic fungi isolated from cucumber plants and fruits, namely: Fusarium acuminatum, Botrytis cinerea and Aspergillus niger, were used. Only one strain -Bacillus sp. Q10, allowed the same mycelial growth as in control variants (Table 1) and showed no inhibitory effect to any of the tested fungi (Table 2). Mycelial growth and growth inhibition of F. acuminatum and B. cinerea caused by Pseudomonas spp. application are shown in Figure 1 (a and b, respectively). The strain P. chlororaphis Q16 had earlier been tested for antifungal activity against fungal pathogens of several medicinal plants and showed good biocontrol potential (Jošić et al., 2012a(Jošić et al., ,b,c, 2015. In this study, the strain was used for comparison with the more recently isolated strains Pseudomonas sp. N3, K24, K27 and K35. P. chlororaphis Q16 inhibited in vitro mycelial growth of F. acuminatum by 45-50%, B. cinerea by 55-60% and A. niger by 32-37%, depending on bacterial culture or fraction applied (Table 2). Similar values were observed for the Pseudomonas sp. K27 strain. The Pseudomonas sp. N3 strain inhibited only B. cinerea by about 40%. The strain Pseudomonas sp. K35 was almost as effective as the strain K24, with inhibition values from 68-80%, compared to 70-80% for K24. These two strains inhibited the mycelial growth of all fungi regardless of culture or fraction applied. The lowest significant differences of leaf weight and plant mass were observed for the mixture M4 at 95% probability level. The lowest significant differences in leaf weight were found for M5.
After two months of growth of cucumber seedlings, several parameters influenced by different combinations of Pseudomonas spp. and Bacillus sp. Q10, except plant height, were not all statistically different from the control.
Chlorophyll and NBI in all tested combinations of Pseudomonas spp. and Bacillus sp. Q10 were at the level of untreated control (Table 4). dIScuSSIon Production of quality and healthy nursery is a significant precondition for high-quality production in greenhouses and in open fields. Restricted use of pesticides and mineral fertilizers is a global tendency that creates preconditions for conservation of soils and ecosystems in general (Turan et al., 2014). Therefore, application of plant growth promoting bacterial strains is an environmentally safe control method in nursery and plant production.
In this investigation, the effects of Pseudomonas spp. strains with different antifungal activities and Bacillus sp. Q10 strain with PGP activity were tested on cucumber plants. The detected phytopatogenic fungi showed the same growth rate in the control as in treatment with the strain Bacillus sp. Q10. No inhibitory effect on the mycelial growth of F. acuminatum and Aspergillus niger was observed when Pseudomonas sp. N3 strain was used in dual culture. The strain P. chlororaphis Q16 showed lower antifungal activity against fungal pathogens of cucumber than some medicinal plants (Josic et al., 2012 b). Two Pseudomonas strains (K24 and K35) were selected for our pot experiment for their high antifungal activity against all tested fungi that reached 67% and 82%, respectively. All tested fractions -filtrate of supernatant and HSAF, as well as the whole culture of bacteria -were almost equally effective in mycelial growth suppression.
F. acuminatum is a toxic species that infects many plant species. This pathogen has been found on cucumbers in Turkey, on cereal crops in Spain (Marín et al., 2012) and on shallot onions in Georgia (Parkunan & Ji, 2013). Elimination of this pathogen from fields and greenhouses is essential for growing healthy plants. P. chlororaphis Q16 inhibited mycelial growth of this fungus 43-52%, which was a lower efficacy than those achieved by Pseudomonas spp. K35, which reached 67-75%, and K24 -82%, the maximum of F. acuminatum growth inhibition.
P. chlororaphis Q16 in vitro inhibited the mycelial growth of B. cinerea 55-60%. We chose the isolates Pseudomonas spp. K35 and K24 for further investigation of biological control of B. cinerea because of their inhibition rates of 74% and 82%. In future investigation, a planned field trial will include P. chlororaphis Q16 because of the production of several antibiotics (Jošić et al, a,c, 2015 already known as effective at suppressing plant pathogens and related diseases. Microbial agents, such as Serratia plymuthica (Azami-Sardooei, 2011) or plant activators such as chitosans (Ben-Shalom et al., 2003), have been reported as important and safe control methods of the grey mould disease caused by B. cinerea on cucumber plants.
P. chlororaphis Q16 in vitro inhibited mycelial growth of A. niger 32-37%, and the applied bacterial culture and fractions achieved similar values. P. chlororaphis Q16 had earlier been shown to cause strong inhibitory or even fungicidal effects against the  (Jošić et al., 2015). The inhibition zones in this study ranged from 18-22 mm, which is similar to the values in previous reports, where the inhibition zones of A. niger in dual culture with P. aeruginosa were 14 mm (Kishore et al., 2005(Kishore et al., , 2006 and 18 mm (Khanuchiya et al., 2012), while it was 17 mm with P. fluorescence (Khanuchiya et al., 2012).
Better results in mycelial growth inhibition of A. niger were obtained using Pseudomonas sp. K35, with inhibition zone values from 37-40 mm, while the best inhibition was observed using Pseudomonas sp. K24, which showed 41-45 mm inhibition zones. These results are in concordance with data previosly reported by Lukkania and Surendranatha Reddya (2014) for different fluorescence Pseudomonas spp. inhibiting A. niger growth and forming inhibition zones of 22.6-55.8 mm.
Our data showed which combination of bacteria had the most favourable effect on cucumber plant growth, and proved a suppressive effect against its phytopathogenic fungi. Different mixtures of strains did not react at the same time or in the same way. Growth dynamics (plant height) differed depending on the applied strain of Pseudomonas sp. Treatment M4 -mixed Bacillus sp. Q10 and Pseudomonas sp. K24, which ultimately achieved the highest plant height (41.91cm), reached in the first height measurement (8.05) a value only slightly higher than the control (16.4 cm). The first treatment of plants with Bacillus sp. Q10 mixed with different Pseudomonas spp. (two days after sowing) had the greatest impact on initial seedling growth. Treatment M3 -Bacillus sp. Q10 mixed with P. chlororaphis Q16, stimulated initial growth (17.98 cm) but ultimately had the lowest effect (35.42cm) in the last measurement (1.06). This can be attributed to the effectiveness of P. chlororaphis Q16 in stimulating the early phases of development of germinating seeds. In contrast to M3 treatment, the strain Pseudomonas sp. K24, which is a constituent of the mixture M4, lent that treatment its strongest impact in the later phases of seedling development, which resulted in maximal seedling height.
Bacterial suspension should be applied during the growth phase when it has the greatest impact, i.e. when enzyme activity is the strongest. Similar results of growth in morphological traits that directly or indirectly impact the yield were found in studies conducted by Dursun et al., (2010), Yolcu et al., (2011), andMoshabaki Isfahani andBesharati, (2012).
We recommend the suspensions M4 and M5 because they had the greatest efficiency regarding the morphological parameters of seedlings, and had the highest level of statistically significant differences against the control, especially for stimulation of the final phase of growth before transplanting. For stimulation of the initial phases of development, the suspension M3 is recommended, which is very important for early production of seedlings for growing in plastic greenhouses, when it is hard to achieve an optimal growing regime, especially temperature.
Comparing the effects of the tested mixtures of bacterial strains against the control we found the chlorophyll content and nitrogen balance index to be at the control level. The result was expected because the seedlings were grown on substrates containing sufficient nutritional matter and the nutritional needs of plants in the control were fully satisfied. A stimulating effect of PGP bacteria was therefore not observed. Our results were not in consistence with data from other studies (Moshabaki Isfahani & Besharati, 2012;Dursun et al., 2010;Yolcu et al., 2011) in which significant differences were detected regarding chlorophyll contents between treated cucumber plants and the control. High levels of chlorophyll influenced by PGPR in vegetable production have been reported for cabbage (increase in plant diameter, height, chlorophyll content of up to 47.5%), (Turan et al., 2014) and some other cultivated plants (Ghazvini et al., 2014). It was found that some soil bacteria have inhibitory action, but strains that increase the level of cytokines were also found, which resulted in better growth, increased level of chlorophyll content, weight of fresh fruit and size of cotyledon leaves of cucumber compared to plants not treated with PGPR (Hussain & Hasnain, 2009). In this study, an absence of negative effects of Bacillus sp. Q10, P. chlororaphis Q16 and Pseudomonas sp. K35 on morphological and physiological parameters of plants was confirmed, as well as a statistically significant increase in plant height when exposed to Bacillus sp. Q10 and Pseudomonas sp. K24. This combination can even have a protective effect on plants, through the presence of strain K24, which is particularly efficient in suppressing phytopathogenic fungi isolated from cucumber and other plants (unpublished data). Further research on poorer substrates that are used in cucumber seedling production is still to prove the expression of PGP traits. Based on all relevant results, an adequate formulation of bacterial mixtures will be made for promoting the growth of cucumber plants in the best way. Krastavac (Cucumis sativus L) je važan predstavnik familije Cucurbitaceae, a proizvodnja zdravog rasada je nephodna za visoku produktivnost u plastenicima i na otvorenom polju. Da bi se umanjila primena pesticida i mineralnih đubriva i pri tome sačuvalo zemljište, ispitan je uticaj bakterija stimulatora biljnog rasta (PGPB) na rast biljaka i zaštitu od patogena. Primenjeni su sojevi Pseudomonas spp. sa različitim antimikoznim delovanjem i Bacillus sp. soj Q10 sa PGP aktivnošću. Praćena je antagonistička aktivnost Pseudomonas spp. na fitopatogene gljive izolovane sa biljaka krastavca: F. acuminatum, B. cinerea i A. niger. Testiran je uticaj prekonoćne kulture bakterija, supernatanta i termostabilnih antifungalnih faktora na rast ovih fitopatogena. Pseudomonas sp. K35 i K24 sojevi, koji pokazuju 70-80% inhibicije rasta gljiva bez obzira na primenjenu kulturu ili frakciju, efektivniji su od P. chlororaphis Q16 i Pseudomonas sp. K27. Sojevi Pseudomonas spp., koji su ispoljili visok stepen antagonizma, kombinovani su sa sojem Bacillus sp. Q10 i procenjen je uticaj na rast i zdravstveno stanje biljaka krastavca. U zavisnosti od primenjenog soja Pseudomonas sp., razlikovala se dinamika rasta krastavca. Tretman M3 -kombinacija Bacillus sp. Q10 i P. chlororaphis Q16 uticala je na početnu fazu porasta biljaka, dok je treatman M4 -kombinacija Bacillus sp. Q10 i Pseudomonas sp. K24, imala najviše uticaja na visinu biljke na kraju merenja. Uočene su značajne razlike za masu lista i masu biljke (M4) i masu lista (M5 sa sojem K35), dok sadržaj hlorofila i nivo NBI nisu imali značajne razlike kod svih ispitivanih kombinacija. Dobijeni rezultati ukazuju da je M3 pogodan za rane faze razvoja biljke, a kombinacije M4 i M5 pogodne su za zaštitu biljaka i u kasnijim fazama porasta biljaka. Potpuna ekspresija PGP svojstava za M4 i M5 može se utvrditi tek posle testiranja na siromašnim supstratima, koji će biti upoređeni sa rezultaima dobijenim na supstratima sa dovoljno hranljivih materija.