Effect of Ulvan on the Biocontrol Activity of Debaryomyces hansenii and Stenotrophomonas rhizophila against Fruit Rot of Cucumis melo L

In the present study, the following was investigated: (a) The effect of ulvan on in vivo and in vitro biocontrol of Debaryomyces hansenii and Stenotrophomonas rhizophila against Fusarium proliferaum and (b) the effect of ulvan on in vivo and in vitro growth of D. hansenii and S. rhizophila and muskmelon quality parameters. The results showed that the biocontrol activity of D. hansenii and S. rhizophila could be enhanced by ulvan (5 g/L). The combination of ulvan and S. rhizophila resulted in a more effective control of fruit rot in comparison to fungicide benomyl. On in vitro growth of F. proliferatum, individual treatments of D. hansenii and S. rhizophila inhibited spore germination and mycelial growth with no statistical difference with the combined treatments. Ulvan does not have a direct effect on the in vivo and in vitro growth of D. hansenii and S. rhizophila. Furthermore, the combined treatments improve the natural disease incidence and quality parameters like weight, firmness, total soluble solids (TSS), and pH. These results suggest that the use of ulvan may be an effective method to improve the biological activity of D. hansenii and S. rhizophila.


Introduction
Muskmelon fruit (Cucumis melo L.) is commercialized worldwide because of its flavor and nutritional content [1]. During fruit ripening, it is easily perishable and susceptible to fungal pathogens during storage, transportation, and commercialization [2]. Fruit rot caused by Fusarium spp. is one of the most serious diseases of melon fruit, and it is generally controlled by applying synthetic fungicides [3]. However, indiscriminate use of synthetic fungicides causes environmental problems, puts humans at risk, and may proliferate fungicide resistance [4].
Biological control of postharvest disease is an effective and nonchemical alternative. It relies on the use of antagonist microorganisms which limit or stop the development of fungal pathogens [5]. In previous studies, the yeast Debaryomyces hansenii and the bacteria Stenotrophomonas rhizophila have showed good results and promissory characteristics as biological control agents. D. hansenii and S. rhizophila have significantly inhibited pathogens like Aspergillus spp., Fusarium spp., Colletotrichum spp.,

Fruit
Muskmelon (Cucumis melo L. var. reticulatus) fruit were collected from a commercial orchard located in El Pescadero, Baja California Sur, México. Fruit of uniform size at commercial maturity stage were selected and transported immediately to the laboratory. Fruit without physical damage or symptoms of fruit rot were disinfected with 2% (v/v) sodium hypochlorite for 2 min, washed with sterile distilled water, air dried at room temperature, and placed in plastic containers prior to use.

Pathogen Inoculum
The pathogen Fusarium proliferatum was previously isolated from infected melon fruits and maintained in potato dextrose agar (PDA, at dose of 39 g L −1 ) plates at 4 • C for storage [26]. To reactivate the culture and verify their pathogenicity, the pathogen was inoculated into wounded melon fruits and re-isolated onto PDA after infection was established. Spore suspension was obtained from 10 days old cultures PDA at 25 • C, and spore concentration was determined using a hemocytometer and adjusted to 10 4 spores/mL with sterile distilled water prior to use. The antagonist microorganisms were obtained from the Centro de Investigaciones Biológicas del Noroeste (CIBNOR), La Paz Baja California Sur, México, and were originally isolated from the Ojo de Liebre hyperhyaline lagoon (27 • 35 and 27 • 52 north latitude and 113 • 58 and 114 • 0 west latitude). D. hansenii and S. rhizophila were maintained in PDA and tripticase soy agar (TSA, at dose of 40 g L −1 ) plates respectively at 4 • C for storage. Liquid cultures of D. hansenii and S. rhizophila were grown in 250 mL Erlenmeyer flasks containing 50 mL of potato dextrose broth (PDB, at dose of 39 g L −1 ) and tripticase soy broth (TSB, at dose of 40 g L −1 ), respectively; both microorganisms had been inoculated with a loop of each culture and were incubated on a rotary shaker at 180 rpm and 27 • C. D. hansenii concentration was adjusted to 1 × 10 6 CFU mL −1 with a hemocytometer and the cells suspension of S. rhizophila was adjusted to 1 × 10 8 CFU mL −1 using a UV/V spectrophotometer (HACH, Dusseldorf, Germany) at 660 nm and absorbance of 1.

Chemical Treatments
Ulvan (OligoTech ® , Elicityl Ltd., Crolles, France) solution was prepared at 5 g L −1 using sterile deionized water. The synthetic fungicide used in this study was benomyl at 1000 ppm and synthetic bactericide bactrol at 500 ppm.

Effect on Mycelial Growth
The effects of ulvan, D. hansenii, and S. rhizophila on the mycelial growth of F. proliferatum were assessed as described by Zhou et al. [27]. A 10 mm diameter hole was made in a 90 mm diameter plate containing 20 mL of nutrient broth (NB, at dose of 31 g/L). As treatments, 100 µL of: (1) 1 × 10 6 CFU mL −1 D. hansenii; (2) 1 × 10 8 CFU mL −1 S. rhizophila; (3) 5 g L −1 ulvan; (4) 1 × 10 6 CFU mL −1 D. hansenii + 5 g L −1 ulvan; (5) 1 × 10 8 CFU mL −1 S. rhizophila + 5 g L −1 ulvan; (6) sterile distilled water (control); and (7) 1000 ppm benomyl were deposited into the holes. After 2 h, 100 µL 1 × 10 4 spores mL −1 suspension of F. proliferatum was deposited into each hole. The plates were incubated at 27 • C for 7 days. The mycelial radial growth was measured with the ImageJ ® program, which measured the relative area of the calibration parameter and the lesion site in pixels and converted the measurement of lesion site in mm 2 based on the known value of the calibration. Inhibition percentage (I%) was calculated by the following equation: I% = (Gc − Gt)/Gc × 100, in which Gc means the radial growth of the pathogen in the control treatments and Gt means the radial growth of the pathogen with the treatments. Each treatment was replicated ten times.

Effect of Ulvan on the Growth of D. hansenii and S. rhizophila In Vitro
The experiment was conducted in petri plates (90 mm) of PDA for D. hansenii and TSA for S. rhizophila (20 mL per plate). One hundred milliliters of: 1 × 10 6 CFU mL −1 D. hansenii; or 1 × 10 8 CFU mL −1 S. rhizophila was spread with an ethanol-flamed glass rod. After 2 h, 4 equidistant holes (10 mm diameter) were made and 100 mL of: (1) 5 g L −1 ulvan for both microorganisms, (2) 1000 ppm benomyl for D. hansenii, or (3) 500 ppm bactrol for S. rhizophila were deposited in 4 holes. Sterile distilled water was used as control. The plates were incubated at 27 • C for 2 days. The number of CFU was determined by colony density and inhibition percentage (I%) was calculated by the following equation: I% = (Ga−Gu)/Gc × 100, in which Ga means the CFU of the antagonist in the control treatments and Gu means the CFU of the antagonist with chemicals. Each treatment was replicated ten times.

Determination of Quality Parameters
The weight loss (%), fruit firmness (N), total soluble solids (%), and pH were measured to evaluate the effect of ulvan, D. hansenii, and S. rhizophila on quality parameters. For weight loss, fruit from plastic containers were weighed before and after storage. Firmness values were determined by compression after application of a load of 9.8 N using the GY-texture Analyzer at two opposite sides of the equatorial region of the fruit. A homogeneous sample of fruit was prepared by crushing it in the mortar and pestle and a few drops of fruit juice were used for the total soluble solids (TSS) and pH. TSS was determined with a digital Abbe refractometer (PR-32, Atago Co., Tokyo, Japan) at room temperature. A few drops of the fruit juice were placed on the refractometer for measurement of total soluble solids percentage.

Statistical Analysis
To assess the advantage of in vivo combined postharvest treatments (BCAs + ulvan) with respect to the same treatments applied alone (BCAs or ulvan), the type of interaction (additive, synergistic, or antagonistic) was evaluated. The synergy factor (SF) was calculated according to the Abbott's formula [29]: SF = EO/EE; where EO and EE are, respectively, the observed and expected biocontrol percentage (C%) of the combination. EE was calculated as follows: (Ea + Eb) − (Ea × Eb/100), where Ea = C% of postharvest treatment (BCAs); Eb = C% of postharvest treatment b (ulvan). If SF = 1, the interaction between the combination treatments was identified as additive; if SF < 1, the interaction was antagonistic, and if SF > 1, the interaction was synergistic.
The data were processed by one-way analysis of variance (ANOVA). Statistical data analyses were performed using the software program STATISTICA 10.0, and the post hoc least significant difference Fisher test (p ≤ 0.05) was used for comparison of the means. When it was necessary, data were transformed into arcsine square root values to normalize distribution before analysis of variance.

Effect of Ulvan, D. hansenii, and S. rhizophila on Muskmelon Fruit Rot by F. proliferatum
D. hansenii, S. rhizophila, and ulvan had a significant effect on disease control of fruit rot caused by F. proliferatum on muskmelon fruit stored at 27 • C for 7 days (Table 1). While ulvan treatment had a slight effect on disease control with 14.3%, in combination with S. rhizophila, it had the most effective disease control and was significant similar to benomyl treatment (64.3%). According to Abbott's formula [29], the treatments D. hansenii + ulvan and S. rhizophila + ulvan had synergistic effect in comparison with their individual treatments. On lesion diameter (Figure 1), single treatment of S. rhizophila was better than D. hansenii + ulvan treatment. S. rhizophila + ulvan were the most effective treatment on reducing lesion diameter, but not better than benomyl. These results suggest that ulvan enhances the biocontrol activity of D. hansenii and S. rhizophila against fruit rot caused by F. proliferatum in muskmelon fruit.

Effect of D. hansenii, S. rhizophila, and Ulvan on Mycelial Growth and Spore Germination of F. proliferatum
On PDA media, ulvan had no inhibiting effect on the growth of F. proliferatum compared with the control (Figure 2a). Single treatments of D. hansenii and S. rhizophila significantly inhibited the growth of F. proliferatum in vitro. However, D. hansenii and S. rhizophila in combination with ulvan did not differ from single treatments with BCAs. The results on spore germination inhibition showed a similar pattern to mycelial growth inhibition against F. proliferatum (Figure 2b). These results suggest that ulvan does not have a direct effect on mycelial growth and spore germination inhibition of F. proliferatum. On PDA media, ulvan had no inhibiting effect on the growth of F. proliferatum compared with the control (Figure 2a). Single treatments of D. hansenii and S. rhizophila significantly inhibited the growth of F. proliferatum in vitro. However, D. hansenii and S. rhizophila in combination with ulvan did not differ from single treatments with BCAs. The results on spore germination inhibition showed a similar pattern to mycelial growth inhibition against F. proliferatum (Figure 2b). These results suggest that ulvan does not have a direct effect on mycelial growth and spore germination inhibition of F. proliferatum.

Effect of D. hansenii, S. rhizophila, and Ulvan on Mycelial Growth and Spore Germination of F. proliferatum
On PDA media, ulvan had no inhibiting effect on the growth of F. proliferatum compared with the control (Figure 2a). Single treatments of D. hansenii and S. rhizophila significantly inhibited the growth of F. proliferatum in vitro. However, D. hansenii and S. rhizophila in combination with ulvan did not differ from single treatments with BCAs. The results on spore germination inhibition showed a similar pattern to mycelial growth inhibition against F. proliferatum (Figure 2b). These results suggest that ulvan does not have a direct effect on mycelial growth and spore germination inhibition of F. proliferatum.

Effect of Ulvan on the in Vivo Population Dynamics or in the in Vitro Growth of D. hansenii and S. rhizophila
Ulvan exhibited no significant negative effects both on the in vivo populations ( Figure 3) and on the in vitro growth of D. hansenii and S. rhizophila (Figure 4). Both BCAs grew efficiently on muskmelon tissue during the period of time quantified. After 3 days of incubation, there were significant differences between S. rhizophila and S. rhizophila + ulvan treatments. It was found that for the rest of treatments during the period of incubation, there were no significant differences between single and addition of ulvan on treatments. Chemicals totally inhibited the in vivo growth of BCAs. These results suggest that ulvan does not have a direct effect on the in vivo and in vitro development of both BCAs.

Effect of Ulvan on the In Vivo Population Dynamics or in the In Vitro Growth of D. hansenii and S. rhizophila
Ulvan exhibited no significant negative effects both on the in vivo populations ( Figure 3) and on the in vitro growth of D. hansenii and S. rhizophila (Figure 4). Both BCAs grew efficiently on muskmelon tissue during the period of time quantified. After 3 days of incubation, there were significant differences between S. rhizophila and S. rhizophila + ulvan treatments. It was found that for the rest of treatments during the period of incubation, there were no significant differences between single and addition of ulvan on treatments. Chemicals totally inhibited the in vivo growth of BCAs. These results suggest that ulvan does not have a direct effect on the in vivo and in vitro development of both BCAs.

Effect of Ulvan on the in Vivo Population Dynamics or in the in Vitro Growth of D. hansenii and S. rhizophila
Ulvan exhibited no significant negative effects both on the in vivo populations ( Figure 3) and on the in vitro growth of D. hansenii and S. rhizophila (Figure 4). Both BCAs grew efficiently on muskmelon tissue during the period of time quantified. After 3 days of incubation, there were significant differences between S. rhizophila and S. rhizophila + ulvan treatments. It was found that for the rest of treatments during the period of incubation, there were no significant differences between single and addition of ulvan on treatments. Chemicals totally inhibited the in vivo growth of BCAs. These results suggest that ulvan does not have a direct effect on the in vivo and in vitro development of both BCAs.  means ± standard deviation of ten replicates Different letters are significantly different (p ≤ 0.05) according to Fisher test.

Effect of D. hansenii, S. rhizophila, and Ulvan on Natural Disease Incidence and Quality Parameters
Fruit surfaces were inoculated to explore the preventive activity of D. hansenii and S. rhizophila alone or in combination with ulvan against fruit rot. Even though 70% of the fruits in the control developed decay symptoms after 7 days of storage, the disease incidence (DI) was significantly reduced with all treatments (Table 2). DI under treatment with S. rhizophila + ulvan (13.3%) was statistically similar to treatment with benomyl (10.0%). The combined treatments of BCAs with ulvan were more effective to reduce the disease incidence of muskmelon fruits than the single application. The quality parameters of three fruits from ten replicates were measured (Table 2). Treatment with benomyl and water (control) significantly increased weight loss and decreased the firmness of muskmelon fruit. On total soluble solids (TSS), no significant effects were observed. Single treatment of ulvan or the combination with D. hansenii or S. rhizophila decreased the pH level.

Discussion
The efficacy of biological control alternatives should be comparable to the level of control provided by conventional fungicides in order to obtain general acceptance [30]. Obtaining such high

Effect of D. hansenii, S. rhizophila, and Ulvan on Natural Disease Incidence and Quality Parameters
Fruit surfaces were inoculated to explore the preventive activity of D. hansenii and S. rhizophila alone or in combination with ulvan against fruit rot. Even though 70% of the fruits in the control developed decay symptoms after 7 days of storage, the disease incidence (DI) was significantly reduced with all treatments (Table 2). DI under treatment with S. rhizophila + ulvan (13.3%) was statistically similar to treatment with benomyl (10.0%). The combined treatments of BCAs with ulvan were more effective to reduce the disease incidence of muskmelon fruits than the single application. The quality parameters of three fruits from ten replicates were measured ( Table 2). Treatment with benomyl and water (control) significantly increased weight loss and decreased the firmness of muskmelon fruit. On total soluble solids (TSS), no significant effects were observed. Single treatment of ulvan or the combination with D. hansenii or S. rhizophila decreased the pH level.

Discussion
The efficacy of biological control alternatives should be comparable to the level of control provided by conventional fungicides in order to obtain general acceptance [30]. Obtaining such high levels of control with biological control alternatives is difficult [15]. Thus, there is a tendency to promote an integrated system rather than a single one [31]. The results in the present study demonstrate that the combination of ulvan with D. hansenii or S. rhizophila can result in a significant enhanced control of fruit rot in muskmelon, compared with single treatments of D. hansenii, S. rhizophila, and ulvan ( Figure 1) (Table 1). Previous studies have demonstrated that the biocontrol ability of different antagonist microorganisms to control postharvest diseases of fruits can be significantly improved by combining alternative, but compatible treatments [32]. Chitosan possess antifungal properties and the ability to elicit host defense responses; hence, it has been suggested as an effective additive to improve the biocontrol performance of the antagonistic yeasts Candida saitona and C. laurentii [33]. The combination of NaHCO 3 and the bacterial antagonist Burkholderia spinosa is effective on the suppression of anthracnose, crown rote, and blossom end rot on banana [34].
To understand the direct effect of ulvan alone or in combination with D. hansenii and S. rhizophila on the growth of F. proliferatum, we investigated the effects of ulvan on mycelial growth and spore germination in vitro. In previous studies, ulvan promotes the conidial germination and appressoria formation of Colletotrichum gloeosporioides [35,36]. Nonetheless, our results showed that ulvan did not influence the mycelial growth of F. proliferatum. However, it slightly inhibited the spore germination of F. proliferatum, probably due to chemical PDA alteration (Figure 2). By contrast, single treatments of D. hansenii or S. rhizophila were found to be similar to the combined treatments with ulvan on spore germination inhibition. D. hansenii and S. rhizophila have the capacity to alleviate unfavorable conditions and grow efficiently [11,13]. Both BCAs probably reduce the slight spore germination by inhibition due to chemical alteration of the medium. It has been reported that ulvan did not exhibit any direct fungal activity against Alternaria brassicola, Colletotrichum lindemuthianum, and Uromyces appendiculatus [37][38][39]. These different results might be related to different sensitivities of fungal species to ulvan and dissimilar testing methods might explain these apparently contradictory results on fungal species.
We propose that the efficacy of ulvan in combination with D. hansenii and/or S. rhizophila in controlling fruit rot of muskmelon by F. proliferatum might be linked to the fruit-mediated mechanisms which increase defense response, like priming, PR proteins, and oxidative burst. Jaulneau et al. [40] elucidated that the ulvan-induced defense response on Medicago truncatula is mediated by the jasmonic acid signaling pathway. On wheat and rice, ulvan has a priming effect by increasing the initial oxidative burst and by enhancing the resistance against powdery mildew [41]. Cluzet et al. [24] concluded by a microarray that ulvan increases the expression of codificant genes to phytoalexins, PR proteins, and structural proteins. Although ulvan has a small effect on disease control, its effect is attributed to systemic acquired resistance (SAR) mechanisms and priming, which acts after systemic induced resistance (ISR) mechanisms [24,40,41]. Further investigation needs to be carried out in order to elucidate which mechanisms of resistance are induced in muskmelon fruit by ulvan.
During the last two decades, numerous BCAs have been isolated, identified, and applied to control postharvest decay of different fruits and vegetables [32]. It is crucial for BCAs to colonize fruit tissue more efficiently than the pathogen to compete for space and nutrients [42]. Moreover, our data showed that D. hansenii and S. rhizophila grew rapidly in muskmelon wounds ( Figure 3). However, the direct antifungal activity of BCAs is an important mechanism of action during the colonization time and the efficacy of this mechanism relies on the rapid colonization level of BCAs, thereby inhibiting the early pathogenic process by fungi [42]. Previous studies have shown that D. hansenii significantly inhibited the mycelial growth of Monilinia fruticola (74.4%) and M. fructigena (44.1%), and S. rhizophila significantly inhibited the mycelial growth of C. gloeosporioides (93%) [8,43]. According to previous results, D. hansenii and S. rhizophila control the in vitro growth of F. proliferatum by mechanisms such as mycoparasitism by lytic enzymes and secondary metabolites excretion, e.g., surfactants and volatile organic compounds [44]. In addition, ulvan did not have any influence on the growth of the BCAs in vivo or in vitro (Figures 3 and 4). To our knowledge, this is the first time that ulvan has been applied in combination with BCAs to control fungal diseases of fruit.
It is known that resistance in harvested fruit is associated with levels of senescence, as it drops considerably with the onset of tissue senescence [45]. In a previous study, the effect of combining Pichia membranefaciens and benzo-thiadiazole-7-carbothioic acid S-methyl ester (BTH) on the control of blue mold by Penicillium expansum in peach fruit showed that quality parameters were not impaired [31]. Ulvan in combination with both BCAs decreased natural disease incidence and significantly maintained the fruit firmness and weight (Table 2). An initial increase in the TSS content of fruit may be due to the hydrolysis of insoluble polysaccharide into simple sugars, but subsequently, TSS content decreased as the storage period increased, which is related to a higher respiratory process [46]. In our results, ulvan by the combined treatments with BCAs showed the highest TSS values by probably delaying the respiratory process. During muskmelon fruit maturation, the pH increases from approximately 5.3 to approximately 6.8 [45]. The increase in pH promotes the activity of the poligalacturonase enzyme that is related to the pathogenicity and virulence of F. proliferatum [47,48]. In our results, ulvan and the combined treatments with BCAs maintained the lowest pH values.

Conclusions
The results presented in our study showed that ulvan enhances the effect of D. hansenii and S. rhizophila in controlling fruit rot in muskmelon, but ulvan does not have direct effect on BCA growth. Also elucidated was the effect of ulvan on pathogenicity of F. proliferatum and development of fruit rot disease of muskmelon. The mode of action of both treatments may have complemented each other, whereby ulvan probably provides protection by muskmelon resistance induction, and BCAs inhibit fungal growth colonization by competition for space and direct antifungal activity. However, the mechanism by which ulvan enhanced the biocontrol efficacy of BCAs is complex and may be a result of several different interactions among ulvan, BCAs (D. hansenii or S. rhizophila) pathogen, and fruits. In this study, we found a new methodology to improve the performance of the antagonistic D. hansenii and S. rhizophila for controlling fruit rot in muskmelon during postharvest stage.