ORIGINAL ARTICLE

^ICentro de Biotecnología , Universidad Nacional de Loja. Ciudad Universitaria Guillermo Falconí Espinosa “La Argelia” - PBX: 072547252 - Casilla Letra “S”, Loja-Ecuador. ]]> ^IIAgritechbio, Riobamba - Ecuador.
^IIIFacultad de Ciencias Agropecuarias Universidad Marta Abreu de Las Villas, Santa Clara-Cuba.
^IVDepartamento de Ciencias Naturales. Universidad Técnica Particular de Loja-Ecuador.
^VCarrera de Ingeniería Ambiental, Universidad Estatal Amazónica, km 2 1/2 vía Puyo a Tena (Paso Lateral), CP 160150, Puyo-Ecuador.

Palabras claves: Fusarium, species, babaco, identificación.

INTRODUCTION

The babaco (Vasconcellea heilbornii var. pentagona, Badillo) is an important crop in Ecuador because of the increasing exports of this exotic fruit and its derivatives to the European Union, mainly to Germany and Netherlands with an export volume of 2 351.43 t. This represents more than 4.2 billion dollars, supporting social development in regions where this fruit is grown by small farmers (1, 2). However, diseases are serious limitations to babaco production by causing yield reduction or complete crop loss (3, 4).

One of the most important diseases of babaco is Babaco Vascular Wilt (BVW). The main symptoms of the disease are chlorosis of the lower leaves and rot at the stem base, which leads to epinasty of leaves and plant wilt. Once flaccidity has set in the affected plants, they die within a short period, reducing the production of babaco (3, 5). During the early stages of the pathogen establishment, the growth of babaco seedlings is normal. However, BVW symptoms appear one month after the pathogen infects seedlings. Leaf discoloration usually starts at the bottom of the seedlings and spreads gradually to the top. In the advanced stages of infection, the plant suffers defoliation. BVW disease may occur in both young and old plants. In serious cases the main roots loose their feeder roots, resulting in dieback and eventual death of the seedling (6, 5).

The causal agent of BVW disease is Fusarium oxysporum (3), which was identified and classified as Fusarium oxysporum f sp. vasconcellae (7).  This serious disease causes severe crop losses in most highland provinces in which babaco is grown (1). Fusarium infection reduces the economic lifespan of the plantation from 5 years to 1-3 weeks (3, 8). Affected plants may survive for several weeks, but death occurs frequently in the first week after infection (3).

Molecular methods, which are faster and more sensitive than morphological identification, are also used to identify Fusarium species (12). The use of PCR and DNA sequence analysis of internal transcribed spacer (ITS) regions has become a diagnostic routine for the detection, identification, classification and phylogenetic analysis of many fungi at the species level (13, 14). ITS regions sequences are highly variable in Fusarium, and taxon-selective ITS amplification has been used to detect fungal pathogens such as Fusarium (15, 12).

Nevertheless, according to previous studies, only  Fusarium oxysporum is the agent causing the vascular wilt of babaco (4, 3), but an integral characterization using cultural, morphological, and molecular criteria of this causal agent have not been done yet. Therefore, the aim of this research was to characterize pathogenic isolates of Fusarium spp. associated with babaco morphologically and molecularly.

Samples were collected in areas with the highest production of babaco in the province of Loja from January to March 2013. Each area was georeferenced using a Global Positioning System (GPS). Samples were taken in the cantons Loja (locations: Amable Maria, El Carmelo, Chantaco, Reymincha, Gonzabal, and La Retama) and Saraguro (locations: Cochapamba, Tuncarta, Quisquinchir, and Las Lagunas) (Figure 1).

Nine samples of root rots of babaco were collected from the canton of Loja, which is located between the geographical coordinates 03°39’55” to 04°30’38” south latitude and 79°05’58” to 79°32ʹ48” west longitude, with a height of 2 100 mosl; the temperature varies between 16 and 21°C. Twelve samples of root rots of babaco were taken from the canton of Saraguro. It is located between the geographical coordinates 03°31’38’’ to 04°30’20’’ south latitude 79°43’41’’ to 80°60’30’’, with an altitude between 2 000 to 3 500 mosl; the temperature varies between 15 and 20°C (16).

Plants with chlorosis in the lower leaves and rotting at the stem base were selected. They were cut transversely to check vascular stem discoloration (wine-coloured). Root samples and symptomatic stems were packed in Ziploc bags for further analysis in the plant pathology laboratory at the National University of Loja.

Isolation and identification of Fusarium spp. was performed using the following protocol: roots of plants were washed with tap water until they were free of soil. Subsequently, plant tissue of the affected parts of stems and roots were selected and cut into sections of 0.3 to 0.5 cm length. The samples were disinfected in sodium hypochlorite (NaClO) at 2.5% for two minutes, in 70% alcohol for one minute, and rinsed three times in sterile distilled water for one minute. This procedure aims to eliminate those saprophytes and non-pathogenic microorganisms which are found on the plant tissue surface.

Five sections of the affected plant tissue were selected and placed inside Petri dishes (90 X 15 mm) containing PDA medium (Potato 200 g, Dextrose 20 g, Agar 20 g, distilled sterile water 1000 ml; pH 5.5). The plates were then sealed with Parafilm® and incubated at 28°C for 48 hours at 28°C according to Leslie and Summerell (10). Monosporic isolation was done and the fungi were preserved in mineral oil.

The morphological characterization of isolates of Fusarium spp. was performed with the keys proposed by Barnett and Hunter (17), Watanabe (18), and Leslie and Summerell (10), which consisted in determining various characteristic parameters such as: type of mycelium, colony color (surface and reverse) according to the Chart of  Rayner (19), radial growth rate, and the observation, quantification, and measurement of the reproductive structures.

Where: RGR: Radial growth rate; Fg: Final growth (diameter in mm); Ig: Initial growth (diameter in mm); Et: End time (days); St: Start time (days).

The values of the initial (Ig) and final (Fg) growth and the start (St) and end time (Et) corresponded to the difference between one day of growth and the time elapsed. Isolates were grouped considering the categories of growth: slow (10 mm/day), medium (> 10-12 mm/day), and fast (> 12 mm/day) (21). 

In addition, the measurement and characterization of the reproductive structures (micro and microconidia) were done. Microcultures of each isolate were incubated at 28°C for 72 hours. After the incubation time, the samples were observed using optical microscopy (Olympus Series: 1D81617, Germany).  One hundred (100) microconidia and 100 macroconidia were randomly taken to determine their average size (30). The conidia were counted in each of the isolates by using the quadrant counting method in the Neubauer chamber with a magnification of 40 X for calculating of final concentration of colony forming units ml^-1 (CFU) (22).

Identification of the fungal genus was according to Barnett and Hunter (17), Watanabe (17) Leslie and Summerell (10).

The statistical analysis was performed with the use of the Statgraphic Centurion v.16.1 package. For analysis of RGR, quantification and reproductive structures, measurements were carried out in a completely randomized experimental design with four replications. Differences between isolates were analysed by statistical One Way Anova and Tukey HSD test with a confidence probability of p <0.05.

The DNA extraction was performed from axenic-monosporic cultures, according with the methodology proposed by Rojas-Triviño (22) with slight modifications. Single spore isolates were inoculated in PDB (potato dextrose broth) (two Erlenmeyer flasks 250 ml per isolate), two mycelial discs (5 mm in diameter) with active growth were used for inoculation.

The fungal DNA was extracted using Purelink Plant Extraction Kit Quick Gel Extraction, Invitrogen TM, (Catalogue K1830-01) according to the manufacturer’s specifications. The extraction product was then analysed by gel electrophoresis on 1% agarose (one g of agarose in 100 ml of 1X TBE buffer).

An internal fragment of ITS (Internal Transcribed Spacer) regions was amplified using the specific primers for Fusarium reported by Abd-Elsalam et al. (12): ITS-Fu-Fwd (5`-CAACTCCCAAACCCCTGTGA-3`) e ITS-Fu-Rev (5`-CGACGATTACCAGTAACGA-3`). PCR amplification contained a final volume of 50μL: 34μL MilliQ water, 5 μL of dNTPs (2 mM each), 5 μl of 10X PCR-buffer, 1.5 μl of each primer (ITS-fuf/ITS-fur) 1.8 μl of MgCl2, 0.2 μl Taq DNA polymerase, 1 μl of extracted DNA. Amplifications were performed with a first cycle of 2 min at 95°C for denaturation, 30 cycles of amplification (1 min at 94°C, 30 s at 54°C and 1 min at 72°C), and a final extension at 72°C for 5 min.

All Polymerase Chain Reactions (PCR) were done in a thermocycler (BioRad serial 583BR01218), amplifications were checked by gel electrophoresis on a 0.7% agarose (1 g of agarose in 150 ml of TAE buffer 1X) with 115 V for 45 min. The gel was stained with 1 ml of ethidium bromide (10 mg ml-1) and bands were visualized on a UV transilluminator according to the procedure of Abd-Elsalam et al. (12).

The PCR product was verified by 0.7% agarose gel electrophoresis (band of 400 bp) and the sample was directly purified without removing the gel bands, using a purification kit (Purelink PCR Quick Gel Extraction and Purification, Invitrogen (Catalogue K220001)). Sequencing was performed by capillary electrophoresis and PCR product samples were sent to the Catholic University of Leuven, Belgium, where the sequencing reactions were performed with the same primers used for amplification.

An automatic sequencer Applied Biosystem 3100 was used. Nucleotide sequence assembly and alignment were performed with the Codon Code Aligner program and compared with a search for homologous sequences using the BLAST-N program and algorithm MEGABLAST (23), on the basis data NCBI gene bank (National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov).

Almost 75% of all isolates showed a type copious or floccose mycelium.  According to the radial growth rate  AML1 and COC2 were fast growing isolates, which covered the Petri dish completely before the last evaluation period (Table 2).

Isolates QUI2 (Quisquinchir) and TUN1 (Tuncarta) produced the longest conidia, both isolates belonged to the canton of Saraguro. Therefore, there was variability in this parameter among the isolates obtained (Table 3). A great variability was observed among the twelve isolates, which suggested the possible coexistence of  several Fusarium species in the same lesions.

The twelve isolates associated with MVB showed hyaline, globose, oval or elliptical microconidia. None of the conidia showed septa, and they were 17.08 to 8 36 X 4.60 to 4.0 µm (length x width). In addition, all isolates showed hyaline macroconidia boat shaped, curved or straight pointed at the ends with 65.08 to 37.32 X 8.20 to 5.04 µm (length x width). It should be noted that the great majority of the isolates showed macroconidia without septa, except the isolates ECM 2, REY1, COC 3, and QUI 1, in which 3-4 septa were observed (Table 3).

Based on the morphological and cultural characteristics, the twelve fungal isolates associated with VWB sampled in different agroclimatic zones of Loja province belonged to several species of Fusarium spp. Ochoa and Fonseca (24) reported F. oxysporum isolates from babaco tissues showing a cottony mycelium and a typical pale-pink to purplish discoloration of the species when it sporulated. On the other hand, according to Leslie and Summerell (10), F. oxysporum mycelium produces dark purple or dark magenta pigments when is grown on PDA medium, never clear or whitish yellow. However, the pigmentation observed by Dubey et al.  (21) was violet, yellow and grey. Furthermore, Shahnazi et al. (25) reported F. solani isolates from black pepper producing pigmentation that was cream, white or whitish cream, wine, coffee, black, and grey. In our study, the different isolates of Fusarium produced pigmentation with different colors such as purple, purple or whitish yellow, pink or magenta pink, and white or yellowish white, mainly in the centre of the colony. This result showed a great variability among different isolates of Fusarium associated with babaco vascular wilt.

Ochoa and Fonseca (3) and Irzykowska et al. (26), argued that F. oxysporum was the causal agent of BVW, which had a rich mycelium (cottony or floccose). On the contrary, the descriptions by Dubey et al. (21) assert that F. oxysporum is a predominantly floccose mycelium. Conversely, Lezcano et al. (26) and Shahnazi et al. (24) reported that Fusarium species had an aerial mycelium, and sometimes plush. In our study, the results based on the type of mycelium of Fusarium agreed with those reported by the first three authors above cited; moreover, the results concerning the morphological diversity of the isolates obtained in the present study suggested the presence of various Fusarium species including in the species F. oxysporum.

Variability was also observed among isolates in the daily mycelial growth, which might be caused by nutrient defficiency (5). The macroconidia were less abundant compared with the microconidia. Morphology of these structures varied and the sickle, curved, and straight shapes were the most frequent. Most isolates showed microconidia without septa, except the isolates El Carmelo 2 (ECM 2) and Reymincha (REY 1). The microconidia were globular, oval,  and elliptical to oval. The macro and microconidia size were outside the range informed by Watanabe (17) and Leslie and Summerell (10), with an average of (29,1-45 µm x 2,9-4,7 µm) for F. oxysporum macro and microconidia, which suggested thar other species of the genus might be present (Table 3).

Similarly, Córdova-Albores et al. (11) reported that the morphological characteristics of the species of Fusarium spp. isolated from gladiolus corms were greatly variable. The macroconidia showed varied forms: papillate, blunt, hooked, and foot shaped. Likewise, the microconidia were oval, reniform, pyriform, and globose. The great variability in macroconidia size (87,51 to 82,54 µm x 50,50 to 48,72 µm), with three to four septa, was evident, and the microconidia were non-septate or with one septum. Similar results were observed in the present study.

The twelve isolates associated with BVW identified by their cultural and morphological characteristics were analized for identification by using  molecular techniques  PCR products of approximety 400 bp were obtained (Figure 2). Comparative analysis of sequenced ITS region showed an identity of 99 to 100% with isolates of Fusarium spp. from the Genbank. According to the sequence homology, four species of Fusarium (F. camptoceras, F. temperatum, F. oxysporum, and Fusarium sp.) were identified (Table 4).

Based on this result, Abd-Elsalam et al. (12) used the primers ITS1 and ITS4 to identify F. oxysporum f. sp. vasinfectum, F. oxysporum, Fusarium moniliforme, and F. solani, and they obtained bands with molecular size from 550 to 570 bp,  results which did not agree with the those obtained in the present work. Nevertheless, these authors did not perform sequencing of the PCR products. Similarly, Abd-Elsalam et al. (12) designed the specific primers ITS-Fu-Fwd and Rev ITS-Fu-Rev for a rapid identification of Fusarium.  These primers showed high specificity for amplifying a Fusarium region with a molecular size of 389 bp. In this research, the PCR products obtained with these primers showed an  estimated molecular size of 400 bp (Table 4).

Therefore, of the twelve molecularly characterized isolates, only 11 corresponding to F. camptoceras, F. temperatum, or F. oxysporum could be identified. The remaining isolate showed similarity to align the BLAST 100% and it was identified as Fusarium. sp. It was evident that using the molecular tools used in this research allowed the identification of microorganisms, integrating morphological, cultural, and molecular data. Future studies will help the understanding of the pathogenic mechanisms allowing the development of integrated strategies for a more effective and environmentally friendly management (27).

In the present study, the cultural, morphological and molecular characterization determined showed great variability among species of Fusarium spp. reporting new species of Fusarium (F. camptoceras, F. temperatum, F. oxysporum, and Fusarium sp.) associated with vascular wilt of babaco (BVW) in southern Ecuador.

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Angel Robles-Carrión, Centro de Biotecnología , Universidad Nacional de Loja. Email: anroca1980@gmail.com