Daucus carrota L. - seed germination and natural infection by Fusarium spp. affected by Pseudomonas spp.

SUMMARY The inhibitory effect of indigenous Pseudomonas strains on Fusarium spp. isolated from seeds of a new variety of carrot - ‘Vizija’ and their effects on seed germination were observed. Pseudomonas spp. strains were applied as bacterial suspensions (culture, 10 6 CFU mL -1 ) and cell-free supernatant (CFSa -10 6 and CFSb -10 8 CFU mL -1 ) fractions by sowing seeds during 7 (I) and 14 (II) days of incubation. The germination of control ‘Vizija’ seeds was 19% after the first and 40% after the second incubation period. Pseudomonas chlororaphis Q16 strain exhibited statistically significant increases in seed germination with all applied fractions and incubation periods, showing values of 43-62% and 55.5-91%, i.e. increments of 24-43% and 15.5-51% compared to the control, after 7 and 14 days of incubation, respectively. P. chlororaphis K35 showed lower but significantly different seed germination values (38-67%) for all variants, except for 7 days old culture. Pseudomonas sp. Ek1 had weaker seed germination potential, showing statistically significant increment only for CFSa,b (I) and CFSb (II) fractions. All tested Pseudomonas strains inhibited the growth of three Fusarium species isolated from ‘Vizija’ seeds: F. solani, F. oxysporum and F. subglutinans. Natural infection was observed in 20% (I) and 54% (II) of ‘Vizija’ seeds. P. chlororaphis K35 was the most efficient antifungal strain, reducing seed infection 97.5-100%, followed by Q16 with 95-100%, showing no statistically significant mutual difference. Pseudomonas sp. Ek1 showed a weaker antifungal activity and reduced seed infection by 85-96.75%. The application of P. chlororaphis Q16 and K35 as strains effective in improvement of carrot seed germination and growth inhibition of the seed pathogens F. solani, F. oxysporum and F. subglutinans , can be further tested in carrot production for more beneficial effects.

Daucus carrota L. -seed germination and natural infection by Fusarium spp. affected by Pseudomonas spp.

INTRODUCTION
Over the last few decades, there has been a growing need for chemicals that effectively prevent plant diseases. Due to potential risks to human health, it is necessary to find a biological alternative to chemical control products for plant pests and diseases. Pests and chemicals do not always have long-term effects on plants, even when applied in early stages of plant growth. A lot of attention has been given to bacterial strains as biocontrol agents that are able to reduce disease incidence caused by pathogenic fungi and also to provide a long-term antifungal protection. Microbiological fungal control is important for some agro-economically important crops such as carrots.
Carrot (Daucus carrota L.) is one of the most important vegetables, widely used for human consumption due to its high concentration of nutritional ingredients such as β-carotene. ß-carotene is known for its beneficial effects on human immune system as a precursor of vitamin A. Recently, β-carotene has been examined for its potential anti-cancer properties. Carrots are also rich in other vitamins, proteins, fibers, energy, micro and macro elements, minerals, as well as antioxidants. Carrots have high Fe 2+ , Cr 2+ , K + and Ca 2+ contents, and contain 40 calories per 100 g of dry weight, which is significantly higher in comparison with other vegetables used for human consumption (Hanif et al., 2006). Due to beneficial effects on human health, carrots are widely consumed as a fresh vegetable, as well as its juice, pickles and preserved carrots and candies (Sharma et al., 2011).
Besides the conventional orange carrot, a purple variety of this vegetable is also used for human consumption. The roots of purple carrots are characteristic for their high sugar content. It has been shown that purple carrots have higher concentrations of some important phenolic compounds, such as anthocyanins, in comparison with conventional orange carrots (Zadernowski et al., 2010). In this research, a new variety of purple carrot named 'Vizija' was used as a test-plant. This variety is characterized by a dark purple root exceeding 25 cm in length. 'Vizija' is characterized by a great yield potential, even under unfavorable cultivation conditions.
Carrots are very susceptible to fungal infection caused by Fusarium spp. The genus Fusarium consists of a variety of fungal species that can commonly be found in soil and water. These fungi are known to be plant-pathogenic for a wide array of economically and agriculturally important plants. Some of Fusarium species can produce a wide range of mycotoxins which are harmful for plants, as well as for humans and animals (Moretti, 2009). Fusarium oxysporum can be found in various types of soil worldwide. These fungi have a great economic importance as they can induce root rot diseases, as well as vascular wilts in various crops. During the last few decades, F. oxysporum has attracted a lot of attention of researchers worldwide due to its disease inducing potential in a variety of plant hosts. Zhang et al. (2014) reported the first case of carrot disease caused by F. solani and F. oxysporum in China with disease incidence of up to 80%, causing symptoms such as dry rot lesions and brown cankers. In Serbia, Stanković et al. (2015) observed a carrot disease caused by F. avenaceum inducing similar symptoms, such as crown and root rots. F. subglutinans is another species, commonly causing pitch canker. This species is associated with a wide range of hosts, such as carrot, maize, pineapple, pine, sorghum and mango (Nirenberg, 1976).
Over the last few decades, some of the bacteria in the genus Pseudomonas have been used as microbiological fungal control agents against a wide array of fungal species, including Fusarium. Some of Pseudomonas bacteria have already shown a great potential when used as biocontrol agents against Alternaria solani (El-Sayed et al., 2008). Pseudomonas chlororaphis is most commonly used as a fungal antagonist due to its wide-spectrum of antifungal activity against soil-borne plant pathogens. Some of the most significant antifungal substances produced by P. chlororaphis are hydrophobic compounds, phenazine-1-carboxamine (PCN), chitinases, proteases and hydrogen cyanide (Chin-A- Woeng et al., 2000Woeng et al., , 2005. P. chlororaphis is widely used for plant growth-promoting in the form of inoculants for biofertilization, biocontrol and phytostimulation (Bloemberg & Lugtenberg, 2001).
In this study, the potential of P. chlororaphis and Pseudomonas sp. strains for stimulating seed germination, and their antifungal activity against Fusarium spp. isolated from 'Vizija' seeds was tested.

MATERIAL AND METHODS
The samples of 'Vizija' seeds used in this study originated from the Institute for Vegetable Crops, Smederevska Palanka, Serbia (Figure 1). Pseudomonas sp. (Ek1) and two P. chlororaphis strains (Q16 and K35) used in this research are part of Project III46007 collection of the Institute of Soil Science, Belgrade, Serbia.
Pseudomonas strains were tested in the forms of bacterial suspension (culture) and cell-free supernatant (CFS) for fungal growth inhibition and improvement of seed germination. CFS fractions were obtained by centrifuging bacterial suspension at 13000 rpm for 5 min.
Supernatant was then re-centrifuged in centrifugal filter tubes with 0.22 µm microporous membrane (Millipore). Culture (10 6 CFUmL -1 ) and CFS (CFSa -10 6 and CFSb -10 8 CFUmL -1 ) of each tested strain were applied to seeds by soaking for 30 min. Each fraction of bacterial strains was applied to 100 seeds (25 seeds per each of 4 Petri dishes) in two independent experiments and the results were presented as mean values. Germinability and overall seed health status were examined on filter paper, using the paper towel method. The ratio of seed germination was counted after 7 (I) and 14 (II) days of incubation in the dark at 25°C by counting all germinated and ungerminated seeds.
Three Fusarium species were isolated from 'Vizija' seeds as natural infection agents, and were characterized based on their macroscopic and microscopic morphology as F. solani, F. oxysporum and F. subglutinans. The effect of Pseudomonas strains on Fusarium spp. was determined by counting naturally infected seeds. The results showing the inhibitory effect of Pseudomonas strains were expressed as percentages of reduced infection incidence compared with the untreated control. The data were statistically processed using the Statistica 7 software, one-way ANOVA and Duncan's multiple range test at the significance level of P<0.05.

RESULTS
Stimulation of carrot seed germination by Pseudomonas strains and difference between seed germination induced by each strain and the control sample is shown in Figure  2 and Table 1. P. chlororaphis Q16 induced a statistically significant increment in seed germination for all applied fractions and in both incubation periods. The weakest seed germination potential was observed in Pseudomonas sp. Ek1, showing the same or lower values than control samples when culture or CFSa (II) were applied. * bacterial suspension -culture; cell-free supernatant -CFSa (10 6 CFU mL -1 ) and CFSb (10 8 CFU mL -1 ) **Dissimilar letters in each column mark statistically significant difference (p ≤ 0.05) at the level of 5%, using Duncan's test The CFSb of all strains showed the greatest seed germination potential during both experimental periods, while cultures showed the weakest potential. The seed germination potential of all tested strains gradually increased over time, and germinated seeds ranged from 40% in the control to 91% in the CFSb fraction of Q16 strain. The applied Pseudomonas strains exhibited high antifungal potential against Fusarium spp. The results of seed infection and reduction in infection incidence for all tested strains and application forms are shown in Table 2 and Figure 3. All tested strains showed the best antifungal activity when applied as bacterial suspension; Q16 and K35 strains inhibited infection 100% during the first week. The inhibition of fungal growth was slightly lower during the next seven days for K35 and Q16, although without statistical significance, while Ek1 showed slightly weaker antagonism, and significantly lower values for the two CFS fractions.

DISCUSSION
P. chlororaphis Q16 and K35 were the most efficient strains which significantly reduced natural infection of the carrot variety 'Vizija' with three Fusarium species -F. solani, F. oxysporum and F. subglutinans. The highest seed germination potential was observed in P. chlororaphis Q16, followed by K35, and both significantly stimulated seed germination over time in all applied fractions. Pseudomonas sp. Ek1 was less effective than the two P. chlororaphis strains, showing lower values of seed germination and higher percent of seed infection, but still significantly beneficial compared with the control.
An improvement in germination rate of carrot seeds and efficacy of Pseudomonas spp. as biocontrol agent in control of the pathogen Alternaria radicina using pretreatment with hot water was reported by Lopez-  * bacterial suspension -culture; cell-free supernatant -CFSa (10 6 CFU mL -1 ) and CFSb(10 8 CFU mL -1 ) ** dissimilar letters in each column mark statistically significant difference (p ≤ 0.05) at the level of 5%, using Duncan's test P. chlororaphis is a very strong antifungal agent. Mezaache-Aichour et al. (2016) tested the antifungal effects of P. chlororaphis against F. solani in dual culture, and it showed strong antifungal activity and inhibited fungal growth by approximately 65%. Ghosh et al. (2014) examined the antifungal effects of Pseudomonas spp. against F. subglutinans and found that fungal growth was inhibited up to 95% by Pseudomonas spp. in dual culture. P. chlororaphis decreased the incidence of disease induced by F. solani by almost 50% in cotton plants cultivated in an extremely saline soil under gnotobiotic conditions (Egamberdieva et al., 2015). P. chlororaphis is proved to be one of the most efficient bacterial antagonists against F. solani in controlling tomato root rot in greenhouses (Postma et al., 2013). Chin-A-Woeng et al. (2000) reported that root colonization by P. chlororaphis under gnotobiotic sand conditions is one of the most important steps in bacterial biocontrol of F. oxysporum. Tziros et al. (2007) studied the antagonistic effects of some bacterial strains on F. oxysporum in control of watermelon wilt disease, and found that P. chlororaphis reduced disease severity by 41%, especially during the early stages of disease development, under gnotobiotic conditions. A combined application of P. fluorescens and P. chlororaphis showed a strong effect against the same pathogenic fungus. The authors suggested that P. chlororaphis could be used as a potentially strong agent against F. oxysporum infections.
P. chlororaphis Q16 reduced the disease incidence caused by Alternaria tenuissima up to 86.5% on basil (Ocimum basilicum L., Lamiaceae) under gnotobiotic conditions (Jošić et al., 2012a). Under the same conditions, this strain exhibited a strong antifungal effect on A. tenuissima and reduced disease incidence on cardoon (Cynara cardunculus L., Asteraceae) by 93%. In addition to their PCR detection of a gene for phenazine-1-carboxylic acid (PCA), the authors measured significant amounts of PCA and 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) (Jošić et al., 2012b). The harbouring genes for phenazine antibiotics and their production are among the properties responsible for the strong antifungal effects of P. chlororaphis Q16 strain.

CONCLUSION
P. chlororaphis Q16 and K35 strains tested in this study improved seed germination (19-51%, depending on the applied culture/fraction) and inhibited by 95-100% the fungal growth of carrot seed pathogens, such as F. solani, F. oxysporum and F. subglutinans.
Pseudomonas sp. Ek1 showed 85-96.75% inhibition of fungal pathogen incidence. In addition to the effects of P. chlororaphis strains as antifungal agents and seed germination inducers, field trials are necessary to confirm all their beneficial effects and biocontrol potential in carrot production.