Sampling

In the months of October and November 2016, root samples were collected in avocado trees (Persea americana Mill. var. Hass) under inoculum pressure that presented characteristic symptomatology of descending death known as avocado sadness. The collection site was the experimental garden of the National Institute of Forestry, Agriculture and Livestock Research (INIFAP) located in San Juan Nuevo Parangaricutiro, Michoacán, whose semi-warm, sub-humid climatic conditions with rain in summer, range from 1 200 to 1 600 mm and temperatures of 10 to 28 °C (^{García, 1981}). A directed sampling was carried out close to the drip area at a depth of 30 cm at four equidistant points.

With the help of a straight shovel, the root samples were taken with the presence of damage (necrotic dark brown tissue) of 2 to 6 mm in diameter and placed in polyethylene bags previously labeled with the data from the garden, municipality, georeferencing and agronomic management (conventional or organic), later they were transported to the phytopathology laboratory of the Department of Agricultural Parasitology at the Agrarian Autonomous University Antonio Narro.

Isolates of phytopathogens

The roots were washed with sterile distilled water to fragment them into pieces no larger than 0.5 cm, with the support of a sterile scalpel, a longitudinal cut was made selecting the limits of healthy and diseased tissue, the root cuts were washed in 3% sodium hypochlorite solution for one min, followed by three washes with sterile distilled water and placed on absorbent paper previously sterilized. Once dried, V8^®-PARPH was planted in a selective medium placing four pieces of roots horizontally in 8.5 cm diameter Petri dishes; three boxes per sample, giving a total of 12 roots per tree sampled and finally the isolates were incubated at 28 °C for three days in total darkness (^{Fierro, 2011}).

Purification and multiplication

V8^®-Agar strains with characteristic growth of P. cinnamomi were transferred to culture medium. The purification technique used was by tip of hypha in triplicate, placed in the center of the Petri dish, sealed with cling wrap and incubated at 28 °C in a bioclimatic chamber for 72 h in the Phytopathology laboratory of the Department of Agricultural Parasitology.

Morphological and molecular identification

The pathogens obtained were identified morphologically according to ^{Davison and Ribeiro (1996)} and molecularly through the PCR-ITS technique, extracting DNA according to the methodology of ^{Doyle and Doyle (1990)}; from 0.2 g of mycelium from the pure culture with lysis buffer (50 mM EDTA, pH 8.5, 100 mM Tris HCl, pH 8, 50 mM NaCl, 2% SDS). DNA visualization was performed on a 2% agarose gel stained with GelRed (GenScript^®).

The amplification of the ITS region was carried out with the primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’- TCCTCCGCTTATTGATATGC-3’). In the same way, the product of the reaction was visualized by means of electrophoresis in 2% agarose gel stained with GelRed (GenScript^®) and the PCR product was sent to the phytosanitary diagnostic laboratory UA-LAB.

Evaluation of the growth rate

The culture media were potato-dextrose-agar (PDA), Pea-Agar, Rye-Agar and V8^®-Agar. Three strains with different characteristic growth (cameloid, rosette and stellate) were selected, later for each medium, they were sown in the center of a Petri dish of 8.5 cm in diameter 5 mm diameter explants taken from the margin of a vigorous growth of a colony 8 days after sowing with four repetitions, the boxes were sealed and incubated in bioclimatic chamber where they grew as pure isolation at 28 ±2 °C in total darkness, with the help of a digital vernier, diameter readings were taken they were taken every 24 h at four equidistant points until the last repetition filled the box.

Statistical analysis: once having the measurements of the diameters of each strain in different nutrient medium, linear regression was performed by generalized linear models (GLM) to obtain the parameters of intercept and slope (growth rate), significance of the regression and the value of R² adjusted. The homogeneity of variance test of the errors of the regression models in each species was carried out and the linear coefficients were contrasted with time, using the values of the sum of squares of each regression by means of a test of t (^{Ott and Longnecker , 2010}) using the following expressions:

Where: b₁ and b₂ are the slopes of each regression for each isolation; SS₁ and SS₂ are the sum of squares of each regression model of the equations.

Where: Sp= is the combined estimate of the error of the two regression equations with n-2 degrees of freedom. SME= mean square of the experimental error.

The analyzes were performed using the statistical program R 3.4^®.