Control Alternatives for Damping-Off in Tomato Seedling Production

: In two tomato genotypes, we assessed control alternatives for damping-off with combinations of chemical fungicides and native/commercial strains of biological agents. Forty treatments consisting of 19 levels of mixing products, chemical fungicides, native strains and commercial products from biological control agents, and untreated treatment were used onto Ramsés and Toro hybrids. They were distributed on an incomplete block design in divided plots arrangement, where genotypes constitute the larger ones and the 8-repetition mixed products, the smaller ones. Putting 180 mL of fungal complexes, made of spores and mycellium Fusarium-solani (2 × 106 UFC), Rhizoctonia-solani (1 × 106 UFC), Phytophthora-capsici (1 × 105 UFC) and Sclerotium-rolfsii (mycellium-sclerotia) on each seedling trays, made inoculation possible. The mixtures of (Bacillus spp. + Streptomyces spp. + Trichoderma spp.) + (Propamocarb + Fosetyl); (Bacillus spp. + Streptomyces spp. + Trichoderma spp.) + (Metalaxyl + Chlorothalonil); Pseudomonas fluorescens + Streptomyces + Micromonospore + Sporideamium + Aminoacidos, Péptidos, Carbohidratos) + (Propamocarb + Fosetyl); the native strain of Trichoderma asperellum + (Propamocarb + fosetil) and the native strains Trichoderma asperellum + Bacillus subtilis, diminished damping-off, prevented its appearance and had most significant agronomic characteristics. In contrast to this, combination of (Mancozeb) + (Free iodine) + (Metalaxyl + Chlorothalonil) + (Methyl thiophanate) produced some toxicity in the plant. In addition, Ramsés presented the best agronomic parameters, while Toro had the utmost fresh and dry weight in root.


Introduction
If we consider the number of hectares sown and consequently its production value, tomato (Solanum esculentum L.) is the most cultivated and economically important vegetable worldwide [1]. It occupies a land surface of 5.023.814 ha that contributes to a global production of 177.042.359 t; from which Mexico provides 388.435 t [2]. In recent years, annual production has grown because of an increase in the yield rate and cultivable land [3].
This crop faces numerous phytosanitary issues from early to advanced stages of the production system; the main limiting factors for tomato production are diseases caused by fungi. Seedling seedbeds must be kept protected until they are transplanted into definitive soil. In the southern region, like in other parts of Mexico, the production of tomato seedlings deals with some sanitation difficulties given by these fungi genera: Phytophthora, Sclerotium, Fusarium, Pythium and Rhizoctonia, among others [4]. They are the main soil-borne pathogens that end up causing root/collar rotting and more than 50% of severe damage [5].
Today, most diseases are being controlled with synthetic fungicides, but their indiscriminate usage has had environmental impacts, caused toxicity in men, and induced pathogens' resistance. Consequently, their efficiency lessen due to phytopathogens constant evolution; they increase production costs. Seeking for more efficient and low-cost alternatives for fungi control are being suggested [6].
The tendency to disease control without using chemical products has increased, due to capacity of parasites compete for nutrients or produce compounds opposing to a great variety of fungi genera Rhizoctonia, Fusarium and Pythium [7,8]; for example: the use of beneficial microorganisms as a way to biocontrol diseases [9], especially fungi: Trichoderma, Gliocladium, Coniothyrium and Candida, and bacteria: Bacillus, Streptomyces, Pseudomonas and Agrobacterium [10,11]. The use of isolated strategies had low impact, for that reason, it is very important and necessary to implement integrated management strategies [12]. Based on this, it was put forward to evaluate the effectiveness of damping-off disease management when utilizing, in two commercial tomatoes hybrids, sole third-generation chemical fungicides and a mixing of native/commercial strains from biological control agents.

Site of Study
The investigation was established in a greenhouse dedicated to seedling crops. It is located in the district of Tonatico, Estate of Mexico, that has subtropical climate, an average temperature of 20.5°C a year, and rainfall from June to September with 1199 mm in the year, Latitud 18°80 28" Longitud 99°.67 00" [13].

Factors and Study of Treatments
Two factors were studied: genetic material with the hybrids (Ramsés and Toro) and disease management with chemical fungicides, native strains of fungi or bacteria, and commercial products from biological control agents resulting in 40 treatments (Tab. 1).

Design and Experimental Unit
In 200 cell trays, treatments were distributed on an incomplete block design in split plot arrangement. In big plots, genotypes were arranged as randomized blocks, but in smaller plots, disease management were onto a complete 8-repetition randomized design. The experimental unit consisted of 20 plants per replicate, five of them were randomly selected for variables data. Eight seedling trays were used for each genotype, so that the 19 mixing products and the absolute control with eight replicates could be applied (Tab. 1).

Experiment Setup
Seedling trays were immersed in 0.1% of sodium hypochlorite commercial solution for their sterilization and then put to dry. Subsequently, the substrate used to fill them in consisted of: Sunshine Mix 3, a mix containing 27.2 kg of peat that integrates a uniform combination of moss (Sphagnum canadiense) and added with carbonates to adjust pH (5.9 a 6.2). It was mixed with 13 kg of Vermiculite or peat moss. Finally, it was mixed with 10 kg of Humibac, an organic fertilizer made of bovine manure. And subsequently, trays'cells were completely filled.
Seeds were deposited at a depth of 1.5 cm and covered with vermiculite to keep humidity and allow emergence. Trays were placed in a dark room for four days at 25 ± 1°C. But at the time of emerging, they were moved to a greenhouse for carrying out seedlings management. The growing conditions of the greenhouse were 25 ± 1°C and 70% of relative humidity. Plants were daily irrigated using a finely watering can and fertilized with Hakaphos Violeta in doses of 0.5 g L -1 mixed with irrigation water.

Application in the Management and Inoculation of Treatments for Phytopathogens
After ten days, treatments on seedlings with true leaves, for this, we used the recommended average dose (Tab. 1). Two days after the mixing of products was applied, a suspension of spores was inoculated with concentrations of Fusarium solani: 1.8 × 10 6 UFC, Rhizoctonia solani: 1.0 × 10 6 UFC, Phytophthora capsici: 1 × 10 5 UFC. But to Sclerotium rolfsii, a mycelium suspension and one thousand sclerotia was prepared. We put on each tray, 180 mL of the fungi complex directly on plants' stalks. Biological commercial products are formulated as powders and the natives strains spores are developed in rice grain.

Variables of Study
Were quantified twenty days after phytopathogens inoculated: seedlings height, number of leaves, dry weight of root, disease severity, disease necrosis, and presence of damping-off. For the evaluation of severity and necrosis, the scale proposed by St-Arnaud et al. [15], was used. The values of the scale range from 0 to 5.
Statistical procedures were performed using the statistical software SAS (version 9.1; SAS Institute, Cary, NC), to analysis of variance (ANOVA) and Tukey followed by a least significant difference (LSD) test (p = 0.05).

Figure 1: Plant height (cm) based on management and genotypes
In a greenhouse [16,17], assessed Trichoderma in vitro strains as bio-controller agents of R. solani and S. rolfsii. T. asperellum species exercised better control over phytopathogens. In the same way [18,19], studied the bio-controlling effect of various species of Trichoderma, and reported that T. asperellum was the most effective in controlling R. solani, as against RIDOMIL GOLD ® (Metalaxyl+Chlorothalonil) fungicide. Hu et al. [20], point out that Propamocarb and Fosetyl have good curative and protective effects against oomycetes, as found in P. capsici, that's being back up in the current investigation. In synthetic fungicides with the best disease management efficiency were (RIDOMIL GOLD ® (Metalaxyl + Chlorothalonil) + PREVICUR ® ENERGY (Propamocarb + Fosetyl)), Matheron & Porchas [21] proved that Fosetyl suppress sporangium formation, motility, zoospores and clamidospores germination; and has a very high efficacy activity, just like with Metalaxyl [22]. It also reduces Phytophthora spp destructive ability. T. asperellum species have been utilized with success in managing soil-borne phytopathogens (R. solani, Fusarium spp. P. capsici & S. rolfsii), either in greenhouses or in the field [23,7,24]. Besides, the T. asperulum native strain represents an alternative to control diseases.
Ming et al. [25] state that Bacillus subtilis holds great potential as bio-controller because of the production of secondary metabolites with antifungal activity. Just the same, Zhu et al. [26] mention that Streptomyces species produce prime chitinolytic enzymes that are responsible for the excellent control over phytopathogens of fungi. In this trial, the PHC ® BIOPAK-F ® commercial product, formed with different strains of Bacillus and Streptomyces, exerted good greenhouse control. It has been reported that Fosetil-al holds a great systematic activity and their efficacy against oomycetes is highly superior which generates great control; and Metalaxyl too [20].

Dry Weight of Root
Toro hybrid showed the average of dry weight in root with 0.64 g (Fig. 2), which was superior to the one in Ramsés (P > 0.0001). Average values ranged from 9 to 88 mg (P > 0.0001). The mix of T12 (Bacillus spp. + Streptomyces spp. + Trichoderma spp.) + (Propamocarb + Fosetyl) had the greater biomass of dehydrated root exceeding the withness by 50%.
The microorganisms included in PHC BIOPACK-F ® are efficient due to bacteria as they exceptionally suppressed the development of phytopathogenic fungi. The species belonging to the Bacillus genus form endospores tolerant to external climate changes [27]. Just the same, Streptomyces have proven that the production of antifungal compounds, both in vitro and field trials, are capable of inhibit S. rolfsii and improve vegetative growth [28]. Moreover, the potential of Trichoderma spp. provides protection against Phytophthora spp and induce resistance in plants as a defense mechanism to Trichoderma colonization [29].

Disease Severity
Genotypes presented similar severities, for instance; Ramsés hybrid has in average of 1.55 to the utilized scale (P > 0.2144) and Toro with 1.46 (Fig. 3). In the management, the mix of (Mancozeb) + (Free iodine) + (Metalaxyl + Chlorothalonil) + (Methyl thiophanate) (T9) exhibit a valor to the 4.0 of the utilized scale (P > 0.0001); which indicated the severe damages done to the stalk by fungi action. We found, low severity in eleven combinations with values of 1.1 having good protection. We support the efficiencies of the commercial biological product PHC BIOPACK-F, for containing gram positive bacteria (Bacillus and Streptomyces spp.), which produce spores resistant to heat and desiccation that can be made as powder, and are compatible to other products [30]. Trichoderma spp., secrete various compounds that induce systematic resistance in plants, among them there's ethylene and jasmonic/salicylic acids [31].

Disease Necrosis
Ramsés hybrid show higher average of necrosis with 2.08 to the utilized scale (P > 0.0059), than Toro hybrid (Fig. 4). As a result of the mixtures and their interactions, Tukey test separated by levels, from which (Mancozeb) + (Free iodine) + (Metalaxyl+Chlorothalonil) + (Methyl thiophanate) (T9) and native strain of Trichoderma asperellum + Cymoxanil (T18) were not efficient since they presented greater necrotic lesions with a valor of 4.5 to the utilized scale. Therefore, mixtures with biological agents were efficient with values of 1.0 (Fig. 4), from which the control achieved 1.5 with some damage in the root 1 to 3%. In this study, the commercial product of PREVICUR ® ENERGY (Propamocarb+Fosetyl), exerted better control with healthy roots. These results coincide to what was found in other studies; they mention this product can efficiently control Phytophthora spp. [32]. Streptomyces spp., are a group of microorganisms being taken advantage of in the production of secondary metabolites and enzymes of commercially importance for biological control of diseases and for other agricultural purposes [33]. It has been described that species of Trichoderma secrete extracellular compounds like gliotoxins, antibiotics, and enzymes for being effective in pathogens elimination [34].

Damping-off Presence
Ramsés hybrid was the most susceptible because the disease appeared after 0.87 days (P > 0.0002), while Toro delay it after 1.25 days (Fig. 5). In regards to management, it should be said that the combinations of products in treatments T12, T13, T14, T16, T17, and T19 were effective because there was no presence of the disease (P > 0.0001). From those that did present disease, Tukey test separated the levels, where the mixture of (Azoxistrobin) + (Methyl thiophanate) + (Bacillus subtilis strain ULTAQ-07) (T10) exhibited earlier symptoms after 0.19 days. But the Trichoderma asperellum + Cymoxanil (T18) native strain was the most tardive with an average of 2.69 days after fungi inoculation (Fig. 5). The β-glucanase and cellulase of B. subtilis and B. licheniformis are substances of deterrent activation, as they hydrolized P. capsici cell wall. Both strains produce siderophore iron chelators in the soil, resulting in the depletion of iron, which blocks pathogens to grow [35]. B. subtilis have proven their effectiveness in controlling Rhizoctonia and Fusarium [36]. There are several isolated antifungal actives known that have been characterized from Streptomyces spp. [37]. This backs up the efficient usage of Streptomyces in the phytopathogens biocontrol [32]. All previous characteristics, make these biocontrol agents reliable and their efficacy is maximized if they are used as mixture. A commercial alternative is PHC BIOPACK-F and TRAZAK, as well as the T. asperellum native strain. These were used for this trial conducted in Cocula, Gro., with efficient results, achieving seedlings vigorius and healthy [38].
Ramsés hybrid presented better plant height, number of leaves, and collar diameter; but Toro achieved greater values of dry weight in root. As compared to Toro, Ramsés showed the highest rates for severity and necrosis, however; the disease appeared first in the Toro hybrid.