Biological material

Bacterial strain Bacillus sp. A8a, which was isolated from the rhizospheric soil of an avocado tree with Phytophthora wilt symptoms in an orchard in Huatusco, Veracruz (^{Méndez­Bravo et al., 2018}), was used as a potential biocontrol agent. The pathogenic agent used in this study was the strain TGR­1­5 of P. cinnamomi, donated by the Plant Pathology Laboratory of the Instituto de Investigaciones Agropecuarias y Forestales of the Universidad Michoacana de San Nicolás de Hidalgo- and isolated from rotting avocado roots in an orchard in Tinguindín, Michoacán. Its virulence was determined in tests performed on Nicotiana benthamiana leaves (^{Mondragón­Flores, 2022}).

Preparation of bacterial inoculant and of the P. cinnamomi zoospore suspension

To produce the bacterial inoculant, the strain Bacillus sp. A8a was streaked on Luria Bertani (LB) agar medium and incubated for 24 h at 26 °C. After this time, a loopful of bacterial biomass was suspended in sterile water. The concentration of the inoculant was adjusted to approximately 1.5 × 108 CFU mL­1, according to the 0.5 standard of the McFarland scale.

The P. cinnamomi zoospore suspension was prepared as described by ^{Zentmyer and Chen (1969}) with slight modifications. Briefly, the P. cinnamomi TGR 1­5 strain was cultivated in Petri dishes with cornmeal agar for six days at 25 °C. Later, mycelial squares of approximately 0.5 cm2 were cut, evenly distributed into two new Petri dishes and 10 mL of clarified V8 liquid culture medium (0.01 g of CaCO3, 10 mL of Campbell’s® V8 juice and 990 mL of distilled water) and incubated for 24 h at 25 °C. After this time, the V8 medium was discarded, the mycelium was washed with 10 mL of Zentmyer solution (1.64 g of Ca(NO3)2, 0.05 g KNO3 and 0.48 g of MgSO4 in 1 L of distilled water) and incubated for 30 min at room temperature under fluorescent light. The wash procedure with mineral solution was repeated six times. The mycelium was then incubated at 23 °C with exposure to fluorescent light for 10 h, followed by 8 h in the dark. To promote the release of zoospores, the salt solution was removed and the mycelium was washed with 10 mL of distilled water at 4 °C. Subsequently, water was added again and the mycelium - water mixture was incubated for 20 min at 4 °C. After this time, the water with P. cinammomi zoospores was recovered. Finally, the zoospores were stained with lactophenol blue (2 drops in 750 mL of solution) to be able to count them in a Nuebauer chamber and the concentration of the suspension was adjusted to 2.5 × 104 zoospores mL­1.

Implementation of the greenhouse experiment

To evaluate the potential of Bacillus sp. A8a as a biocontrol agent against P. cinnamomi, 90 avocado plants of the Méndez variety, of approximately two years­ old, were acquired from the “Piedras Blancas” nursery (Lagunillas, Michoacán, Mexico). The experiment consisted of four treatments: 1) 30 “control” plants, treated only with water [C]; 2) 30 plants infected with P. cinnamomi [Pc]; 3) 15 plants inoculated with Bacillus sp. A8a [B]; and 4) 15 plants infected with P. cinnamomi and inoculated with Bacillus sp. A8a [BPc] (^{Reverchon et al., 2023}). On day 1, defined as the first day of the experiment (t0), half of the plants (n=45) were infected with the P. cinnamomi zoospore suspension, as described by ^{Aveling (1999}) and ^{Masikane et al. (2019}). First, the plants were retrieved from their grow bags and the excess soil was removed from their roots. Later, their root system was submerged for two hours in a suspension with a concentration of 4.9 × 102 zoospores mL­1, which was prepared by diluting 1.6 L of the starting zoospore suspension in 80 L of drinking water. The roots from the other half of the plants were submerged in 80 L of drinking water with 1.6 L of distilled water (non­infected plants). Each plant was transplanted into a 10 L black plastic bag, with a substrate mixture of peat (PRO­MIX®)­perlite­vermiculite (ratio 3:1:1, v/v/v) sterilized at 121 °C for 1 h, with 1 volume of plant nursery soil. After 48 h, the plants were placed under waterlogging conditions (substrate saturation) for five days to favor the infectious process of P. cinnamomi. Next, the excess water was drained and plants were watered with drinking water throughout the experiment to maintain the humidity of the substrate. Bacillus sp. A8a was inoculated 15 days before the infection of the plants with P. cinnamomi. For this, 30 plants were inoculated with 250 mL of the Bacillus sp. A8a suspension, applied on the base of the stem. Fifteen of these plants were assigned to treatment [B], and the other 15 plants, to treatment [BPc]. The bacterial suspension was applied again on the day after the infection (day 2) and once again 15 days later (day 16). The avocado plants were distributed randomly over a metal bed in the greenhouse of the Escuela Nacional de Estudios Superiores (National School for Higher Studies) of the UNAM - Morelia. The experiment was carried out in March and April, 2021, at an average temperature of 31 °C recorded at 11:00 am.

Measurement of morphological and physiological variables in avocado plants

The measurement of morphological and physiological variables was carried out at days 1 (t0), 4 (t1),7 (t2), 13 (t3) and 25 (t4) after inoculation (dai) with P. cinnamomi. The morphological variables that were recorded were height (cm), diameter (cm), and number of leaves, each measured from six individuals for treatments C and Pc, at each time; for treatments B and BPc, measurements were taken from three individuals. The percentage of wilting of each plant was calculated using the following formula:

as reported by ^{Wang et al. (2006}) and ^{Azil et al. (2021}).

For physiological variables, three plants were measured per treatment. Stomatal conductance (mol CO2/m2/s), photosynthetic rate (µmol/m2/s) and transpiration (mmol/m2/s) were measured using the portable gas exchange system Li­Cor 6400 (LI­COR, Lincoln, NE, USA). The measurements were performed on two extended leaves with no apparent damage per selected plant, between 8:00 h and 11:00 h, with a light intensity of 1500 µmol/m2/s and environmental CO2. The water potential (MPa) of the plant was measured at pre­dawn (5:00 h) and at mid­day (14:00 h) with a Scholander 1000 pressure chamber (PMS Instrument Co., Albany, OR, EE.UU.), in two leaves per plant, which were covered with aluminum foil half an hour before measuring to allow the water potential of the leaf and of the stem to become homogenized (^{Aguilar­Romero et al., 2017}).

Histochemical analyses of stem sections and determination of the presence of callose and tannins

To determine callose and tannins in the avocado stems at the end of the infection process (25 dai), a 1.5 cm­long fragment of stem was cut from the basal part of three plants per treatment using pruning shears. The fragments were processed following the methodology described in ^{Johansen (1940}). The fragments were fixed for 24 h in an FAA solution (70 % alcohol, formaldehyde and distilled water in a 5:5:90) ratio. After the fixation period, the FAA solution was removed and an ethanol-glycerol solution was added (1:1, v/v) to preserve the samples. Later, the samples were washed with tap water, dehydrated in increasing concentrations of ethyl alcohol (30, 50 and 70 %) and were passed through a series of increasing concentrations of tert­butyl alcohol (^{Sandoval, 2005}). The stem fragments were placed in histological paraffin (Hycel) and 15-20 µm-thick cuts were made on the transverse and radial faces, using a rotary microtome (Leica RM2125 RTS). The sections were stained with a combination of 1 % tannic acid (in water); 3 % ferric chloride (in water) and 3 % lacmoid (in a 50 % ethanol and 50 % water solution), according to ^{Gurr (1965}). The sections were then washed with a 1% sodium bicarbonate solution (in 50 % ethanol), dehydrated in gradual concentrations of alcohol at 70, 80, 90 and 100 % and the tissues were clarified with absolute xylene. Finally, the sections were mounted on synthetic resin and observed under a compound microscope (Leica DM750M). The tannins were quantified using images captured with a 20× lens in bright-field microscopy. Images were taken of the transversal sections of avocado stems of the four treatments at 25 dai. The obtained images were analyzed using the Software ImageJ (https://imagej.nih.gov/ij/index.html).

Statistical analyses

The results were analyzed in RStudio, v. 2022.12.0. Data normality was verified using the Shapiro­Wilk test and the variance homogeneity, with the Bartlett test. The data obtained from the measurements of morphological and physiological variables, as well as from the quantification of the content of tannins, were analyzed using two­way and one­way analyses of variance, respectively, followed by Tukey’s test for normally distributed data. The data that did not exhibit a normal distribution were analyzed using a Kruskal­Wallis test and a Wilcoxon rank­sum test with a Bonferroni-Holm adjustment. Results were considered significant with a P ≤ 0.05.

Changes in the morpho-physiological variables of avocado plants in response to the inoculation of Bacillus sp. A8a and infection by P. cinnamomi

Our results show that the inoculation of Bacillus sp. A8a in plants infected with P. cinnamomi (treatment BPc) reduced the percentage of wilting by up to 49 % at 25 dai, in comparison to infected plants that were not inoculated with the bacteria (treatment Pc) (Figure 1). In addition, wilting in plants infected with P. cinnamomi increased with time, going from 2.9 % (1 dai) to 50 % at the end of the experiment (25 dai). In contrast, the percentage of wilting decreased in infected plants inoculated with Bacillus sp. A8a throughout the experiment, going from 40.4

% at 13 dai to 7.1 % at 25 dai (Figure 1A). Nevertheless, these differences were not significant, most likely due to the intra-treatment variation.

No significant differences were observed in plant height and stem diameter between treatments for a given time, nor in the interaction between time and treatment (Figures 1B, C). Moreover, no significant differences were detected in the stomatal conductance, photosynthetic rate and transpiration between treatments, regardless of the time of measurement (Table 1). However, the water potential at 25 dai was significantly lower in plants infected with P. cinnamomi (treatments Pc and BPc) than in non­infected plants (treatments C and B), both in their measurement at 5:00 h (Figure 2A) and at 14:00 h (Figure 2B). No significant differences were observed between treatments Pc and BPc in terms of water potential (Figure 2).

Accumulation of tannins in avocado stems in response to the infection by P. cinnamomi and to the inoculation of Bacillus sp. A8a

The percentage of the area covered by tannins at 25 dai was quantified. The infection caused by P. cinnamomi tripled the area of tannins in the inner and outer xylem, compared to the control plants (Table 2). In contrast, the inoculation of Bacillus sp. A8a induced a greater accumulation of tannins in the cortex, although not significantly. The inoculation of Bacillus sp. A8a doubled the accumulation of tannins in the cortex of the uninfected plants (37.5 % in treatment B), and induced an increase of 16.9 % in the area of tannins in the cortex of infected plants (treatment BPc), in comparison with infected plants that were not inoculated with the bacteria. In addition, in infected plants, the inoculation of strain A8a reduced the accumulation of tannins in the inner and outer xylem (Table 2; Figure 3), displaying a shift in the accumulation pattern of tannins induced by the biocontrol strain. Finally, callose was not found in any of the analyzed tissues.

Figure 1 Effect of the inoculation of Bacillus sp. A8a and P. cinnamomi in avocado trees at 1, 4, 7, 13 and 25 dai (days after infection). A) Percentage of wilting caused by P. cinnamomi; B) tree height (cm); C) stem diameter (cm). Bars show the average of the data (n = 6, treatments C and Pc; n = 3, treatments B and BPc) ± s.d. Different letters indicate significant differences (two-way ANOVA, P ≤ 0.05). In Figure 1A, (+) indicates significant differences between times, compared with (-), for the Pc treatment (ANOVA, P ≤ 0.05). D) Representative photographs of the aerial parts and root systems of avocado trees at 25 dai, in the four treatments. C: control; Pc: infected with P. cinnamomi; B: inoculated with Bacillus sp. A8a; BPc: infected with P. cinnamomi and inoculated with Bacillus sp. A8a.