Mexico is one of the most important diversity centers of the Cucurbitaceae family, which includes 118 genus and 825 species, which have had a very important role in culture and economy among the different social stratus. The pipiana pumkpin (Cucurbita argyrosperma Huber) is an economically important crop in Guerrero state, south of Mexico. During 2011, 5,742 ha were grown (SIAP, 2013), and just in the northern part of the state, over 20 family businesses producing the typical food “green mole” are located. The pumkpin seed is the main raw material for making this type of food, as well as the main ingredient in making typical candies such as “palanqueta”, and “jamoncillos”. Guerrero is one of the main seed-producer states nationwide. In some regions of the state this crop is grown in heavy, clay, flat and bad drained soils. During the summer the weather is warm and there is high relative humidity which, in combination with the habit of creeping growth and undetermined crop, it generates a micro-weather with favorable conditions for the development and infection of phytopathogenic fungi inhabiting the soil, which has not been formally identified, and which causes fruit rot, decrease in efficiency and in producers’ economical incomes (Ayvar-Serna et al., 2007). In the productive zone of Cocula, Guerrero, Mexico, severe fruit rot symptoms in pumpkin fruits have been observed. In the region of study and related to pumpkin do not exist information about the diseases management based on biological agents or chemical control. However, in other crops there are several studies of biocontrol for the same pathogens associated with pumpkin rot fruits, for instance the use of Trichoderma asperellum against Sclerotium rolfsii in onion (Guzmán-Valle et al., 2014), Rhizoctonia solani in rice (Oryza sativa L.) (Chen et al., 2015), Phytophthora capsici in pepper (Segarra et al., 2013), as well as Bacillus subtilis and Streptomyces spp., against S. rolfsii, R. solani y P. capsici (Rodríguez-Villarreal et al., 2014; Khabbaz et al., 2015). Similarly, related to the control with chemical products, Gisi and Sierotzki (2008) reports the use of propamocarb, fosetyl-Al, metalaxyl, chlorothalonil against oomycetes; Mendoza-Zamora (1990) applied Benomyl and quintozene (PCNB) against R. solani and S. rolfsii; while Bokshi et al. (2007) reports the use of iodine in the control of true fungi. Because of what has been mentioned, the objectives of this study were (i) to identify the agents causing rot by morphological, molecular, and pathogenic analysis, and (ii) to assess chemical and biological products for the control of the pathogens involved.
Sample collecting
Experimental site
During August and September 2012, pipiana pumpkin fruits showing different symptoms and rot levels were collected from the experimental fielFd of Colegio Superior Agropecuario del Estado de Guerrero (CSAEGro), located in the county of Cocula, north of Guerrero state, Mexico, at 18° 19’ NL and 99° 39’ WL, at 640 m above sea level (masl). The weather is Aw0, which belongs to warm - sub humid, with rains in the summer, and an annual average temperature of 26.4°C, an average of the coldest month (December) is 23.4°C. The temperature oscillation from one month to another is 5 to 7°C. The annual average precipitation is 767 mm (García, 2005).
Vegetal material
The sample size and kind of sampling were carried out under the methodology proposed by Pedroza-Sandoval (2009), by a systematic sampling of transect in W, collecting 10 fruits 30 days after the starting of the fruiting, of the creole genotype “Apipilulco”. The symptoms considered were: caved watery injuries color brown; dry brown spongy putrefactions with a white halo; watery caved spots with a white mycelium growth in the inferior and superior part of the fruit; fruits with nest-type white cottony mycelium (where the fruit is in contact with the ground) and with sclerotia (Zitter et al., 2004).
Isolation and purification
There were some 1 cm2 tissue cuts performed to the fruits showing rot signs and symptoms. These cuts were obtained from the transition zone between the healthy tissue and the sick tissue. The tissue fragments were sanitized using sodium hypochlorite at 1.5% for two minutes; they were rinsed three times with sterile distilled water and then were laid on drying paper for two minutes. One hundred tissue samples were grown, each 5 of them were placed in Petri boxes with growing medium potato-dextrose-agar (PDA) and agar-vegetable juice (V8-Agar). The boxes were incubated at 24°C under continuous black light conditions. The most frequent fungi colonies were transferred to Petri boxes in PDA and V8-Agar growing medium. Mono-zoosporic and hypha tip cultures were obtained according to the methodology described by Fernández-Herrera et al. (2013).
Morphological identification
Out of the fruit re-isolated pathogens, pure cultures were obtained and preparations with glycerol from the asexual reproduction structures (sporangium) and mycelium were done. The preparations were analyzed in light microscopy at 400X. In this analysis, the size, septa, shape of sporangium and hyphae were registered. The identification was performed by comparing the morphological structures of each fungus, following the keys of Sneh et al. (1991), Barnett and Hunter (1998) and Gallegly and Hong (2008).
Pathogenicity test
The isolated fungus were inoculated in 20 fruits of the creole genotype Apipilulco. The fruits were previously decontaminated with sodium hypochlorite at 1.5% for two minutes and then were rinsed with sterile distilled water, letting them dry for 5 min. Four fruits were used for each isolation. The inoculant of each fungus was adjusted at 4×106 zoospores mL-1 for isolation 1; and at 8×105 and 5×106 colony forming units (CFU) mL-1 for isolations 2 and 3, using a Neubahuer chamber. Each fruit was inoculated through spraying 2.5 ml and adding 2 ml of surfactant Tween 20° per liter of sterile distilled water were added. The suspension was applied using a manual sprayer over the pumpkin fruits until runoff point. Four fruits were used as control, which were sprayed using only sterile distilled water. All the fruits were stored in a humid chamber for seven days at 22 ± 2°C and with 100% relative humidity. Once the inoculated fungus showed the same symptomatology than the initially collected tissues, re-isolates were obtained out of the inoculated organism, following the methodology described by Núñez-Rios et al. (2013). The pathogenicity test was performed twice.
Molecular identification
For the DNA extraction and performance of PCR two representative isolates of each fungus were used. For isolation 1, a mono-zoosporic culture was obtained (Quesada et al., 2011), from which five 1-centimeter-diameter discs were cut and put into Petri boxes with 20 ml of sterilized water. The pathogen (oomycete) was incubated at 25 ± 1°C and after four days, the isolations that had produced sporangia were changed at 12°C for 15 min for releasing zoospores. Out of the zoospores solution, one drop was taken and poured on a plate full with growing medium V-8 Agar. It was distributed uniformly and incubated at 25 ± 1°C in a dark place. It was observed 24 and 48 h later in order to detect germinated zoospores (Gallegly and Hong, 2008). Some isolated colonies were taken in order to get the mono zoosporic cultures. Finally, for isolations 2 and 3, a culture of hypha tip was obtained (Okubara et al., 2008; Le et al., 2012). Out of the fungi developed in PDA growing medium, a portion was taken including both medium and mycelium. It was then put into a test tube along with 20 ml sterile water, and shaken strenuously. Afterwards, some serial dilutions with 5 dilution orders were performed. One drop out of dilutions 10-4 and 10-5 were put into boxes with PDA medium, uniformly distributed and incubated at 25°C for 24 h. The fungal growth was cut and re-isolated again in PDA for one week (Fernández-Herrera et al., 2013).
The DNA extraction was done from 50 to 100 mg of mycelium for each isolation, using the DNeasy Plant Mini KitTM, following the manufacturer´s procedure (Qiagen, 2012). The procedure was performed 4 times for the oomycete and each fungus. Universal PCR reactions were performed with ITS-1fu 5’-tccgtaggtgaacctgcgg-3’ and ITS-4 5’-tcctccgcttattgatatgc-3’ primers (White et al., 1990), which amplify two internal intergenic spacers (IGS) and the gene 5.8S of the ribosomal RNA, generating a product between 500 and 900 bases pairs (bp).
This practice was carried out with a reaction mixture in a 25 µl volume, whose final components were: 1X, reaction buffer, 2 mM MgCl2, 200 nM dNTP’s of each one, 20 pmoles of each primer, and one unit of Taq DNA polymerase (Promega). The thermal program consisted in keeping a temperature of 94°C for 2 min, followed by 35 cycles at a 94-55-72°C temperature for 30-30-60 s, and a final extension of 5 min at 72°C temperature. The products of the PCR reactions were separated through electrophoresis in agarose gels at 1.5%, and the bands were observed in an ultraviolet light trans-illuminator UVP brand. The amplified fragments by PCR were directly sequenced and the results were compared against the available sequences in the Genbank of the National Center for Biotechnology Information (NCBI).
Greenhouse control test
For the greenhouse test, unripe pumpkin fruits of genotype Apipilulco were used (directly from the field) weighing approximately 150 g. Six commercial fungicides were assessed for the three pathogens. Two groups were formed based on the type of fungus, the oomycete was assigned to the first group, and the true fungi –deuteromycetes- were assigned into the second group (Table 1). The evaluated variable was the number of days to the pathogen presence (DPP), determining in how many days the colonies would arise on the fruits once the treatments and inoculation of the pathogen were applied. The treatments were applied using a Kwazar manual sprinkler, whose capacity is 1 L. The surface of the fruits were sprinkled using a calibrated quantity of 300 L ha-1 of water. There was a gap of 12 h before the products were allowed inside (FRAC, 2011), then, 2.5 ml of the settled concentration of each pathogen was sprinkled.
Experimental design and data analysis
The greenhouse experiment was established under a completely randomized design with four replicates. The experimental unit consisted of four fruits. The treatments evaluated are described in Table 1. Data were statistically analyzed through analysis of variance and a multiple means comparison procedure by using Tukey’s HSD test with a significance level of 5%. All the statistical analysis were performed using the software Statistical Analysis System (SAS, 2013).
Isolated fungi from fruits with rot symptoms
From the colony developed in PDA and V8-Agar were obtained five isolations. During the sampling in field it was observed that the rot due to the oomycete and one of the deuteromycetes fungus was more common among unripe fruits when there were some conditions like high humidity and precipitation alternated with drought periods. However, the fact that they can harm ripe fruits is not discarded. The second detected fungus was observed when the foliage decreased and there was more light impact on the ground which made the temperature increase as well.
It can harm juicy ripeness fruits but it can also harm unripe fruits. Such results are agreed with what is reported by Zitter et al. (2004).
Morphological identification
Three species were identified: Isolation 1. This fungus showed sporangia 20 - 50 × 15 - 42.5 μm, whit only one papillae and/or with two papillae, of size 6.02 to 7.05 μm wide, and 1.2 to 6.0 μm depth. All the morphological characteristics observed concur with the descriptions by Gallegly and Hong (2008) for P. capsici (Figure 1A and B). Isolation 2. The characteristics were, hyphae 5 to 8 μm wide, hyphae branching out at a right angle position approximately, with a constriction in the branching out joints, close to their place of origin. Another characteristic was sclerotia showing a 1 to 3 mm diameter, typical of R. solani (Sneh et al., 1991) (Figure 1C). Isolation 3. This fungus showed main hyphae with a 4.5 to 8.0 μm diameter, with ring-shape joints which allow secondary and tertiary 2.0 to 4.5 μm-diameter-hyphae arise, and 0.5 to 2.0 mm spherical sclerotia. Due to the characteristics found, it was identified as S. rolfsii (Barnett and Hunter, 1998) (Figure 1D).
Pathogenicity test
Five days after inoculation with a zoospores’ suspension of P. capsici and CFU of R. solani and S. rolfsii, all fruits showed rot, in addition to plentiful fungus mycelium. Among the symptoms presented, there were; caved watery putrefactions with white mycelium growth (Figure 2A); caved watery injuries brown color (Figure 2B); dry spongy putrefactions with a white halo (Figure 2C). The control fruits were free of disease (Figure 2D). P. capsici, R. solani and S. rolfsii were re-isolated from the symptomatic fruits (Figure 2E, F and G).
Molecular identification
When comparing the obtained sequences in the current work against the available sequences in the GenBank, some similarities between 99 and 100% with previously reported sequences were found. This fact corroborates the veracity of the morphological identification. The sequence of the three species found in this current work were deposited in the GenBank under the following accession numbers: KJ652220, for P. capsici; KJ652221, for R. solani, and KJ652222, for Athelia rolfsii (Curzi) C.C. Tu & Kimbr. There was coincidence in the morphological and molecular
identification for the three species. The DNA sequence of P. capsici had a similarity of 99%, with over 100 sequences of the GenBank, but the maximum score (max score) was obtained through two sequences (AJ854286.1 and AJ854287.1) whose strains were isolated in Italy, from Cucurbita pepo L., a specie belonging to the same genus of the pipiana pumkpin in this study.
Greenhouse control test
Days after the pathogen presence
This characteristic is important, since once the pathogen presence has been identified in unripe fruits, these fruits are harmed completely and consequently lost. For P. capsici highly significant differences (P<0.0001) among the treatment effects were found; in the multiple means comparison the active ingredients propamocarb + fosetyl-Al (T5: Previcur® energy), and metalaxyl + Chlorothalonil (T6: Ridomil gold® Bravo) delayed the appearance of P. capsici at day 6, whereas the control treatment was differentiated from all other treatments, and the pathogen appeared 2.82 days after inoculation (Figure 3). For R. solani this variable showed significant differences (P=0.0156) for the treatments applied. T. asperellum (T1), free iodine (T4) and quintozene PCNB (T6) were the best treatments, which delayed the appearance of the pathogen at day 4.67, 4.42, and 4.42, respectively. The control treatment was differentiated from all the products, the pathogen appeared 3.17 days after inoculation (Figure 3). Regarding the S. rolfsii case, high significant differences were detected (P=0.008); the quintozene was the best treatment, it extended the presence of pathogen colonies up to 5.83 days while the T. asperellum and PHC® Biopack did not differentiated each other (Figure 3).
In relation to P. capsici, Hu et al. (2007) point out that propamocarb and fosetyl-Al have a good protective and curative action against oomycetes because they inhibit the oospores production in Phytophthora spp. This has been supported with what has been found in P. capsici in the current research. Additionally, it has been reported that fosetyl-Al has a high degree of systemic activity and efficiency which is generally superior against oomycetes showing a good control (Dufour and Corio, 2013; Gent et al., 2010). It has also been observed that metalaxyl reduces the progress of diseases caused by the oomycetes (Álvarez-Romero et al., 2013). Mihajlovi? et al. (2013) studied different specific fungicides against oomycetes and it was found that fosetyl-Al had a control efficiency of 97.5% over the control of Pythuim aphanidermatum (Edson) Fitzp. The
multi-site fungicide Chlorothalonil turned out to be one of the most effective treatments in the range of fungicides assessed in combination with metalaxyl. This is because it is a highly toxic compound over oomycetes, as Gisi and Sierotzki (2008) report.
Regarding to R. solani, Amrutha et al. (2014) point out that the key factors that contribute to antagonistic effect of Trichoderma are: its quick growth, production of metabolites antimicrobials, and physiological characteristics (El-Katatny and Emam, 2012). Vargas-Hoyos et al. (2012) assessed several isolations of Trichoderma in vitro and in vivo in greenhouses as biocontroller agents of R. solani and S. rolfsii and the specie T. asperellum had the best control over these pathogens. Likewise, Asad et al. (2014) and De França et al. (2014) studied the biocontroller effect of several species of Trichoderma and it was found that T. asperellum was one of the most effective for R. solani control. It is generally accepted that the fungi toxicity of this compound is due to the peroxidation of lipids in the membranes, the effectivity of T. asperellum was confirmed in this research based on the good control obtained. Bokshi et al. (2007) in a study in Cucumis melo L. using iodine against Fusarium sp., Alternaria sp. and Rhizopus sp. to prevent rotting of postharvest fruits, they found that using hot iodine as post-harvest treatment, most of the pathogens causing rot were controlled. Mendoza-Zamora (1990) found that quintozene affected the integrity of the membrane, cell walls and mitochondria of phytopathogenic fungi, decreasing sclerotia and infectious propagules’ formation. Finally, in relation to A. rolfsii, Cavallo et al. (2005) assessed the effect of the PCNB in combination with carboxin+tiram, and they found that such active ingredient was which had more control of true fungi in peanut (Arachis hypogaea L.). Chastagner (2002) reports PCNB as an efficient fungicide for Sclerotium rolfsii Sacc. var. delphinii (Welch) and Rhizoctonia tuliparum Whetzel & Arthur in ornamentals.
Although the previous information supports the results found in this research, it is necessary to continue assessing the active ingredients in the strains of isolated pathogens from pumpkin, to design an integrated management process and to validate it in both greenhouse and field conditions.
The morphological and molecular identification, as well as the pathogenicity tests confirmed that P. capsici, R.
solani, and S. rolfsii were the agents causing the fruits rot in pipiana pumpkin (C. argyrosperma) in Cocula, Guerrero, Mexico. The propamocarb + fosetyl-Al, metalaxyl + Chlorothalonil fungicides, quintozene, and the biocontrol agent T. asperellum delayed the P. capsici, R. solani and S. rolfsii presence at 6.00, 6.00, 4.67, and 5.83 days, respectively, compared to around 3 days for the control treatment.
The authors have not declared any conflict of interest.
This study was made possible thanks to the financial support provided by the Universidad Autonoma Chapingo-135702002 project. The author also wish to thanks Mexico’s National Council of Science and Technology (CONACyT) for the scholarship 329261 to fund his master degree studies. Also we like to thank and highly appreciate the time dedicated by reviewer for patiently correcting, revising, and suggesting improvements on the quality of the manuscript at the different stages of the revision process.
