Plant sampling and fungal isolation

Sampling was directed towards sweet orange trees (Citrus sinensis L.) with symptoms of regressive death and necrosis in the trunk and branches, collected in orchards of the municipalities of General Teran and Montemorelos, Nuevo León, and Llera de Canales, Tamaulipas.

Affected tissues with symptoms of necrosis and regressive death were cut into sections of approximately 1 cm in the transition zone between the necrotic part and the apparently healthy part, disinfected with 10% commercial bleach for 1 min, then washed with water two-distilled, were planted in Petri dishes with potato dextrose agar (PDA) and incubated at 25 ±2 °C with a photoperiod of 12 h light and 12 h of darkness (^{Marques et al., 2013}). The isolated fungi were reseeded by the hypha tip method on water agar and incubated under the same conditions of light and temperature for 4 weeks, in order to obtain pure strains (^{Twizeyimana et al., 2013}).

Morphological and molecular identification of fungi

For the morphological identification of the isolated fungi, cultural characteristics were taken into account, such as growth, color, type and shape of the colony, as well as the growth rate (^{Urbaez et al., 2013}) observed with the microscope in preparations of the fungi stained with lactophenol. The identification of fungal species was carried out using the keys of ^{Phillips et al. (2013)}.

For molecular identification, the strains were grown in PDA at 25 ±2 °C for 7 days. DNA extraction was performed using the DNeasy Plant Mini Kit^TM (Qiagen, Inc.) with slight modifications to the manufacturer’s instructions and the DNA was quantified using a Take3^MR spectrophotometer (Bioselec).

The amplification reactions were carried out in volumes of 25 μL. The ITS-1 region, 5.8S ribosomal gene and ITS-2 were amplified using the oligonucleotides ITS-1fu 5’-tccgtaggtgaacctgcgg-3’ and ITS-4 5’tcctccgcttatttgatatgc-3’ (^{White et al., 1990}).

For the reactions, 5 μL of 5X PCR buffer, 2 μL of MgCl₂ (25 μM), 2 μL of dNTP’s (2.5 mM), 0.2 μL of go-Taq enzyme (5U μL^-1) (Promega^MR), 9.8 μL of Mili-Q grade water, 2 μL of each primer (10 pmol μL^-1) and 2 μL of DNA were used. The PCR consisted of an initial denaturation cycle at 94 °C for 2 min, followed by 35 cycles at 94 °C for 20 s, 55 °C for 25 s and 72 °C for 50 s, followed by a final elongation step for 4 min. The PCR products were visualized on 1% agarose gels, previously stained with 0.5 ng μL^-1 of ethidium bromide and compared with a molecular weight marker (ladder-100^MR, Axygen).

The obtained PCR products were sequenced in the company Macrogen (EE. UU.) and compared with GenBank sequences to confirm the identity of the fungi at the species level.

Evaluation of the pathogenicity of fungi isolated

To determine the pathogenicity of the isolated strains, a bioassay was conducted under greenhouse conditions at the UANL Faculty of Agronomy located in Gral. Escobedo, NL. This municipality is located at 25° 47’ 5.98” north latitude and 100° 17’ 12.12” west longitude at a height of 481 meters above sea level.

For the evaluation of the pathogenicity plants were used sweet orange (C. sinensis) Valencia variety of one year of grafted, which measured 60 to 75 cm in height and were obtained from a commercial nursery. The inoculum was obtained by cultivating the fungi in PDA for 6 days at 25 ±2 °C and 50% relative humidity.

A completely randomized design with 9 treatments was established. Three treatments with the fungi individually, three treatments where the different strains were combined in pairs, a treatment with the combination of the three strains, a control (plants inoculated with sterile PDA) and an absolute control of plants in which only the cut (Table 1).

For each treatment, three repetitions were used.

For the inoculation of the fungi, the plants were subjected to longitudinal wounds of 1 cm using sterile knives. The bark was detached slightly as a flange, causing a wound by plant. In the wound fungi were inoculated by placing mycelial discs of 5 mm in diameter taken from the margin of mycelial growth, then the flange was closed and covered with parafilm to avoid drying the fungus and the plant. Periodic observations were made at seven-day intervals to measure the length of the lesions. Of the plants inoculated with symptoms of necrosis and regressive death, we proceeded to re-isolate and identify the phytopathogenic fungi following the previously described method.

Statistical analysis

An analysis of variance (Anova) was performed to determine the average effectiveness of each treatment and a multiple comparison of means with the Tukey method (p≤ 0.05) with the statistical program SPSS 22 (^{IBM Corp., 2013}).

Isolation, purification and identification of fungi

In the cultures carried out, between 2 and 5 days, several fungi grew, which were purified by hyphae tip and identified by their morphological characteristics as L. theobromae, which in PDA medium initially showed a whitening colony coloration, becoming gray and darkening as the days passed, the conidia initially oval in shape, hyaline and without septa, but as the crop grew older they became septate and acquired a brown coloration. Fomitopsis sp. was identified by its gray-black aerial mycelium and hyaline, unicellular, ovoid and ellipsoidal conidia. E. citricola was identified by its colonial growth of white color, with aerial mycelium and when mature it formed dark and scattered picnidia. The present conidia were hyaline, filiform, unicellular and some curved.

With respect to molecular identification, DNA extraction of the three species of fungi isolated from symptomatic samples of sweet orange was successful. In the 3 cases, an average of 30 ng μL^-1 of DNA with an absorbance ratio of 260/280 between 1.8 and 2 indicating good quality was observed.

In the PCR reactions, the DNA amplification of the 3 fungal species was achieved using the universal primers for fungi ITS-1fu/ITS-4. The consensus sequence of the PCR products was 515 bp for the ITS-1, 5.8S and ITS-2 regions, in addition to partial sequences of 18S and 28S RNA. When comparing the DNA sequences of the isolates with GenBank sequences, in all three cases 99% similarity coincided with L. theobromae (accession JN048466.1), F. meliae (HQ248221.1) and E. citricola (KM396616.1).

Pathogenicity test

Four days after the inoculation, the plants inoculated with F. meliae (treatment 3), showed symptoms of necrosis, wilting and death of branches in descending direction. After 32 days the external necrotic lesions measured between 9.5 and 29 cm long, this fungus being the cause of more aggressive symptoms of descending death (Table 2, Figure 1).

Figure 1 Pathogenicity test of fungi causing regressive death in sweet orange (C. sinensis L.). a) inoculation with mycelium discs; b) symptoms of regressive death; and c) vascular beams of the stem.  

At the end of the bioassay, longitudinal cuts were made of the plants with wilt symptoms, and brown spots on the vascular tissues could be observed in them.

In the treatments in which E. citricola was used as inoculum, the symptoms were less aggressive, since only 1 to 4 cm necrosis was observed around the inoculated area, but they did not express symptoms of wilt.

From all inoculated plants, direct lamellar assemblies and replantings were made in PDA. The morphological and colonial growth characteristics were similar to those previously described for the strains of F. meliae and L. theobromae used for the inoculation. The DNA sequencing of the PCR products coincided in 99% only for L. theobromae and F. meliae, for the re-isolation of E. citricola from the inoculated plants, PCR amplification was not possible and therefore neither DNA sequencing