Morphological and molecular characterization of pathogenic isolates of Fusarium spp . obtained from gladiolus corms and their sensitivity to Jatropha curcas L . oil

The State of Morelos is the third biggest gladiolus producer in Mexico. However, this ornamental is affected by the disease named corm rot or fusarium yellows, characterized by leaf yellowing, epinasty and wilting, and caused by fungi of the genus Fusarium. The first objective was to corroborate the pathogenicity of the 45 isolates obtained. The second objective was to identify and characterize morphologically and molecularly by polymerase chain reaction-internal transcribed spacer (PCR-ITS), the highly pathogenic isolates and comparatively analyze the fungal species involved with the reference strain Fusarium oxysporum f. sp. gladioli (Fog). The third objective was to quantify the phorbol esters in Jatropha curcas oil and evaluate their antifungal potential on mycelial growth and conidial germination of different Fusarium species. Eleven isolates were highly significant pathogenic (P < 0.001). Three fungal species were identified in basal stems and damaged corms taken from field plants, namely F. oxysporum, Fusarium solani and Fusarium proliferatum. Molecular analyzes corroborated the species identified and their sequences were deposited in the National Center for Biotechnology Information (NCBI) gene bank. The percentage of oil obtained was 61.5 %; the phorbol ester content in the oil was 1.52 mg g -1 of 12,13-phorbol myristate. All species identified and the reference strain was sensitive to the 5 mg mL -1 oil concentration.


INTRODUCTION
The gladiolus is one of the main crops in the State of Morelos, which ranks as Mexico's third biggest producer of this flowering plant.Corm rot is caused by Fusarium spp.and results in losses of 60-80% during storage (SAGARPA, 2006).Members of the genus Fusarium are obtained and evaluate their response to J. curcas seed oil.

Sampled material
Corms and plants (yellowing of leaves and corms with basal rot) of five varieties ('White foam', 'Red ewe', 'White ewe', 'Yellow' and 'Sancerri') were collected in the field in seven producing municipalities in the State of Morelos.A total of 34 plants showing symptoms of corm rot were collected from August to October 2011 from commercial fields in Cuautla, Yautepec, Ayala, Tlanepantla, Temixco, Yecapixtla and Totolapan.

Isolation of fungi
Soil and roots were removed from gladiolus corms by washing with running water and then corms were immersed in an acaricide solution (AK ® 20) at a concentration of 15 ml l -1 for 5 min and left to dry.They were disinfected with a sodium hypochlorite solution at 5% for 3 min, rinsed with sterile distilled water and placed in humid chambers for 24 h to promote fungal growth.From mycelium developed in the corms, isolates were made on Potato Dextrose Agar (PDA) medium and incubated at room temperature for 7 days.Monosporic cultures were established as described by Leslie and Summerell (2006).

Pathogenicity tests
Forty-five isolates showed differences in the color of the colonies and initially they were selected as different species.Healthy corms of the variety 'White foam', pre-peeled and pre-treated with a contact fungicide (Captan ® 2 g L -1 ), were planted in sterile soil (121°C, 15 lb pressure for 3 h) under greenhouse conditions.Once the approximately 1-cm stem emerged, 5 mL of the isolated spore solution of each isolate to examine were added at a concentration of 1 × 10 6 spores ml -1 .The reference strain used was Fusarium oxysporum f. sp.gladioli (Fog) already molecularly identified (Garduño-Pizaña et al., 2010) and it was treated in the same way without inoculation.Plants were assessed three weeks after inoculation.

Data analysis
A completely randomized design with 5 replicates for each isolate and a control treatment (reference strain, labeled as Fog) were used.Analysis of variance of repeated means and a multiple comparison against the control were performed with the Holm-Sidak method (P < 0.05) using the Sigma Stat 3.5 statistical package designed by STATCON © Witzenhausen, Germany.

Morphological characterization
For taxonomic identification of the isolates, the classification methodology of Nelson et al. (1983) and Leslie and Summerell (2006) was used.These authors suggest taking as references the colony colors on PDA, and the length of the phialides, chlamydospores, microconidia and macroconidia.The length of the structures was measured using ImageTool © software for Windows Version 3.0 Alpha 4 designed by the Center for Health Sciences, University of Texas.Morphological observations and measurements were made with a compound microscope at 40x.

Molecular characterization
DNA extraction from 12 monosporic isolates was performed by the method described by Doyle and Doyle (1990), with modifications.The quality of the DNA obtained was verified by electrophoresis on 1% agarose gel stained with ethidium bromide (0.1 µg µl -1 ) and observed in a gel documentation system (G Box ® ; Syngene, England).DNA concentration was calculated by measuring the optical density at 260 nm with the following formula:

PCR for ITS primers
The ITS regions were amplified with the primers ITS1 (TCCGTAGGTGAACCTGCGG), ITS2 (TCCTCCGCTTATTGATATGC) and ITS5 (GGAAGTAAAAGTCGTAACAAGG) (White et al., 1990;Haan et al., 2000;Abd-Elsalam et al., 2003;Moreno-Velázquez et al., 2005;Teixeira et al., 2005;González-Pérez et al., 2009), as well as the specific primers for the genus Fusarium reported by Adb-Elsalam et al. (2003): ITS-Fu-Fwd (CGCACGATTACCACTAACGA) and ITS-Fu-Rev (CAACTCCCAAACCCCTGTGA).The primers were synthesized by the Sigma Aldrich ® Company.Each PCR reaction was carried out in a final volume of 25 μl; 5.5 μl of nuclease-free water were mixed with 2.0 μl of DNA of the fungus problem, 2.5 μl of each ITS and 12.5 μl of Taq 2X Master Mix ® (BioLabsInc, New England).The amplification reaction was carried out in a Perkin Elmer ® thermocycler (GeneAmp PCR System 2400) with the following program: initial denaturation at 95°C for 5 min; 35 cycles of denaturation, alignment and extension at 95°C for 1 min and 72°C for 2 min.respectively, and a final extension at 72°C for 10 min.The amplified fragments were verified by electrophoresis in 1% agarose gel stained with ethidium bromide (0.1 μg μl -1 ).The gel was run at 85 V for 40 min and observed in a gel documentation system (G Box ® ; Syngene, England).

Sequencing
The amplified PCR products were purified using the DNA Clean & Concentrator TM -5 Kit (Zymo Research, USA) and analyzed at the Sequencing Laboratory of the Institute of Biotechnology, belonging to the University National Autonomous of México, in Cuernavaca, Morelos.The sequences were aligned and compared with sequences in the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST) (Zhang et al., 2000).

Oil extraction
The extraction of the seed oil was performed according to the Soxhlet method (NMX-F-089-S-1978), with petroleum ether and hexane and 8-h reflux time.About 2 to 5 g of ground J. curcas seeds were weighed and placed in the extraction thimble.After extraction, the solvent was gently evaporated from the flask and the excess petroleum ether or hexane was removed using a BÜCHI model 114 rotary evaporator (BÜCHI ® Labortechnik AG, Switzerland) at 60°C.The calculations to determine the percentage of oil obtained was performed using the formula: Oil (%) = (P-p/M) × 100 Where: P = mass in grams of the flask with the oil; p = mass in grams of the flask; M = mass in grams of the sample.
Phorbol esters were quantified using the high performance liquid chromatography (HPLC) method described by Martínez-Herrera (2006).Specifically, 20 ml of methanol were added to a 2 g sample, which was sonicated for 2 min.The sample was centrifuged at 3600 rpm for 10 min and the supernatant was evaporated almost to dryness in a rotary evaporator (the extraction was repeated 3 times).The standard used was 12,13-phorbol myristate (Sigma Aldrich ® ), which showed a retention time of 25 min.The results were expressed as mg g -1 of sample equivalent to 12,13-phorbol myristate.This analysis was performed in the food laboratory of the Institute of Animal Production in the Tropics and Subtropics at the University of Hohenheim in Stuttgart, Germany.

Mycelial growth
Only the 11 isolates mentioned above and the reference strain (Fog) were used to evaluate this parameter.A 5-mm disc of the pathogen was placed on 60 × 15 mm Petri dishes containing PDA, Tween 20 ® and the oil at different concentrations (2.5, 5 and 10 mg ml -1 ), as well as 4 controls: PDA, petroleum ether (10 mg ml -1 ), Tween 20 ® (10 mg ml -1 ) and Captan ® (2 g L -1 ).They were incubated at 25°C in the dark.Diameter growth was measured daily for 5 days with a Vernier caliper until the control treatments reached the edge of the Petri dish.The experimental design was completely randomized and consisted of 7 treatments with 6 replicates.Analysis of variance (ANOVA) and Tukey's comparison of means test (P < 0.05) were performed using the Sigma Stat 3.5 software program designed by STATCON © Witzenhausen, Germany.

Spore germination
Ten milliliters of sterile distilled water were added to Petri dishes containing the growth of each isolate, then the surface was scraped with a bent metal rod and the filtrate was passed through cotton gauze.Of this suspension, 20 µl were placed on PDA discs of 10 mm in diameter and incubated for 6 to 10 h at room temperature (26°C).Later, a few drops of lactophenol methylene blue were added and the number of germinated spores was determined by photo analysis with Image Tool © software for Windows version 2.01 Alpha 4, developed by the Center for Health Sciences at the University of Texas.Germination was evaluated on nine PDA discs.Mean percentage germination and standard deviations were calculated.

Number of isolates
Their morphological aspect, identified as belonging to the genus Fusarium, obtained a total of 45 isolates.The isolates showed different apparent morphological characteristics, mainly in the color of the culture medium.Nelson et al. (1983) performed a morphological characterization using the coloration on PDA as indicating that each species has a specific color.Based on the coloring obtained, a preliminary classification was conducted, obtaining a total of eight groups as shown in Figure 1.

Morphological characterization
Some of the typical structures of the 11 isolates and the reference strain (Fog) are summarized in Table 1.

Molecular characterization
The DNA had a yield of 220 to 670 µg g -1 of mycelium, the band of the PCR products containing the primers ITS2-ITS5 was 500 base pairs (bp) and the PCR product for the primers ITS-Fu-Fwd and ITS-Fu-Rev was from 410 to 429 bp (Figure 3).For the multiple alignments, the total amplified portion of the 12-nucleotide sequences is shown in Table 2, which corresponds to the complete sequence of both regions (ITS2 and ITS5).In Genbank, the sequence of F. oxysporum f. sp gladioli and isolates T30, T35 and T39 showed a similarity index of 99% with F. oxysporum.Another five isolates showed a similarity index of 99% with F. proliferatum.Isolates T20 and T34 showed no similarity to other sequences (Table 2).

Oil extraction
The oil percentage obtained for both solvents was 61.5%.Phorbol esters in the kernel meal and the oil extracted with petroleum ether were 0.94 and 1.52 mg g -1 , respectively, while the oil extracted with hexane was 0.24 and 0.67 mg g -1 , respectively.

Mycelium growth
Comparison of means of mycelial growth on the last day of each treatment indicated that the eleven Fusarium isolates and the reference strain (Fog) were more sensitive to the concentration of 5 mg ml -1 . Isolates T9, T11, T20, T32, T34, T35 and T40 were also sensitive to the concentration of 2.5 mg ml -1 .For isolate T9, it was observed that the concentration of 5 mg ml -1 showed less mycelial growth than the fungicide Captan ® , while for the isolate T11 incubated at the same concentration (5 mg ml -1 ) and the control with Tween ® 20 there were no significant differences.The isolate most susceptible to the fungicide Captan ® was the reference strain (Fog) (Table 3).

Spore germination
Treatment susceptibility depended on the isolate, as no trend was observed for any specific treatment (Table 4).Isolate T9 presented the lowest germination rate with J. curcas oil at concentrations of 2.5 and 10 mg ml -1 with a percentage germination of 14.40 and 14.43%, respectively, as compared to 98.44% for the PDA treatment.Isolate T24 also showed a low germination rate, which was 12.62 and 17.28% for the concentrations of 2.5 and 5 mg ml -1 , respectively.Isolate T24 was the most susceptible to the treatment with the fungicide Captan ® , presenting a germination of 33.79% while the PDA treatment had 99.77% germination.

DISCUSSION
The highly pathogenic isolates were characterized morphologically, identifying at least 2 species: F. oxysporum and Fusarium solani, which had different morphological features, both gross and microscopic, and which were associated with the afore mentioned species.González-Pérez et al. (2009) reported that these species caused rot in gladiolus in San Martin Texmelucan, Puebla.They also reported that these species differed in terms of colony color and morphological characteristics, coinciding with the results obtained in this work for descriptions of macro-and micro-conidia, phialides and chlamydospores.For their part, Montiel-Gonzalez et al. ( 2005) described the morphological characteristics of F. oxysporum, F. solani, Fusarium lateritium, Fusarium reticulatum, Fusarium equiseti, Fusarium verticillioides, Fusarium culmorum, Fusarium crookwellense, Fusarium proliferatum and Fusarium sporotrichioides present in bean roots in five     content for each solvent, obtained in this study, could be because the two solvents have a different boiling point (68.85 for hexane and 60°C for petroleum ether) and because the phorbol esters in the oil are thermolabile, which means that by applying a higher temperature to obtain the oil with the solvents used, the phorbol esters are degraded.The greatest effect on mycelial growth for the isolates evaluated was with the J. curcas oil treatment at the concentration of 5 mg ml -1 .Growth rates between 0.140 and 0.462 mm day -1 were obtained, when compared with 0.820 to 1.018 mm day -1 for the PDA control.Some authors reported that the effect on the mycelial growth of the compounds present in the essential oils may be due to two factors: the first involves inhibition of extracellular enzyme synthesis, and the second the alteration of the cell wall structure (Tripathi et al., 2009).Siva et al. (2008) evaluated the antifungal effect of aqueous, acetone and ethanol extracts of 20 medicinal plants against F. oxysporum f. sp.melongenae.J. curcas was one of the plants evaluated and showed inhibition percentages of 100% for all extracts used.Donlaporn and Suntornsuk (2010) evaluated the antifungal activity of ethanol extracts of J. curcas seeds and the importance of phorbol esters present in the extracts on F. oxysporum, F. semitectum, Colletotrichum capsici, C. gloeosporioides, Pythium aphanidermatum, Lasiodiplodia theobromae and Curvularia lunata, using concentrations of 0 to 10,000 mg L -1 . Concentrations from 6,000 mg L -1 showed 100% inhibition of mycelial growth for all tested pathogens.These authors are the first to report that phorbol esters are responsible for the fungicidal activity of the extracts, as they note that by removing these compounds from the extracts there were no significant differences as compared to the control.As for the germination of conidia, the three concentrations used in this study were effective for only two isolates (T9 and T24) with germination between 12.62 and 17.28%.Ogbebor and Adekunle (2008) assessed the germination of Drechslera heveae conidia on PDA with J. curcas extracts, attaining a 20% germination rate with the 100% concentration.The three species identified showed symptoms of the disease in the plant, such as leaf yellowing, epinasty and some-times late flowering in the field.It is suggested that in different parts of Morelos, corm rot in gladiolus is caused by three fungal species, which showed morphological, molecular and pathogenic differences, plus different sensitivity to J. curcas oil.All three species were capable of causing the disease.The results of this research demonstrate the antifungal potential of J. curcas oil to control fungi that cause diseases in ornamental plants.

[Figure 1 .
Figure 1.Preliminary classification of isolates according to coloring on PDA medium.

Figure 2 .
Figure 2. Comparison of means against the reference strain Fog (T25) of the pathogenic capacity of isolates of Fusarium spp.Significant difference *P < 0.01; **P < 0.001.

Table 1 .
Morphological characteristics of isolates of the genus Fusarium collected in gladiolus-growing areas of the state of Morelos.

Table 2 .
Morphological and molecular characterization of highly pathogenic isolates from gladiolus corms.
Martínez-Herrera et al. (2010) also agreed with those reported by these authors.Molecular taxonomic results by PCR: ITS confirm the morphological identification of ten isolates, which corresponded to F. oxysporum, F. solani and F. proliferatum.The two isolates that showed no similarity when aligning them using BLAST could be Fusarium species, where genetic variation has occurred due to the indiscriminate use of fungicides in the region.Alternatively, these unidentified isolates could be species not yet reported.The percentage of J. curcas seed oil reported byMartínez-Herrera et al. (2010)coincides with the values obtained in this research in seeds from the state of Chiapas, with a value of 60.4%.The same author reports phorbol ester values of 2.03 mg g -1 in oil and 0.16 mg g -1 in kernel meal.The differences in phorbol ester

Table 3 .
Effect of J. curcas oil on mycelial growth of isolates of Fusarium spp.Reference strain.Means followed by different letters in each column are significantly different by Tukey test at (α 0.05).Values in parenthesis indicate growth rate (mm day -1 ). *

Table 4 .
Effect of J. curcas oil on percentage conidial germination of isolates of Fusarium spp.
*Reference strain.Means and SD.