elocation-id: e3434
Mexico is the leading producer and exporter of avocados in the world. The main producing area is the Central Pacific Region of Mexico (Jalisco, Michoacán, and Nayarit), where 96% of the national production is harvested. Anthracnose in avocado postharvest can reach between 20 and 80% losses due to the deterioration of the quality of the avocado fruit. The objective of the research was to diagnose the causative agent of anthracnose of avocado postharvest in the Central Pacific Region of Mexico, the study was conducted between 2019 and 2020. Completely healthy avocado fruits were sampled and pathogenic fungi that cause anthracnose were isolated, a morphological, molecular, and phylogenetic identification was carried out, as well as their development under fungicide stress. Morphological characteristics indicated that the fungus that causes anthracnose in postharvest belongs to Colletotrichum gloeosporioides sensu lato. Molecular and phylogenetic tests identified the fungi as Colletotrichum siamense, an endophytic fungus capable of existing in two conditions of fruit life (pre- and postharvest). This fungus under fungicide stress remains in an inactive state in response until its effect wears off.
Persea americana Miller, synthetic fungicide, postharvest.
Mexico is the leading producer and exporter of avocados in the world. The main producing area is the Central Pacific Region of Mexico, which includes the states of Jalisco, Michoacán, and Nayarit, where 96% of the national production is harvested (SAGARPA, 2018; SIAP-producción agrícola, 2021). Anthracnose disease in the postharvest stage of avocados can reach between 20 and 80% losses due to the deterioration of the quality of the avocado fruit.
The species complex of C. gloeosporioides is the cause of this disease (Bill et al., 2014), which is characterized by visible symptoms such as black spots on the skin and soft rot in the pulp of immature fruits. During the ripening process, it develops rapidly in the tissues of the pulp and skin until it affects 100% of the fruit (Kimaru et al., 2018). C. gloeosporioides infects the fruit from preharvest (penetration through natural openings, wounds, or direct rupture of the cuticle) and remains dormant (Prusky et al., 2013).
In preharvest, C. gloeosporioides, C. acutatum, C. boninense, C. hymenocallidis, C. siamense and C. tropicale have been identified as causative agents of anthracnose in Michoacán, Nayarit and Hidalgo (Silva-Rojas and Ávila-Quezada, 2011; Jaimes et al., 2015; Campos-Martínez et al., 2016; Trinidad-Angel et al., 2017; Fuentes-Aragón et al., 2018). For the implementation of control methods (chemical or biological), precise identification of the pathogen is required (Fuentes-Aragón et al., 2020a b).
Chemical control at harvest and postharvest is limited to three molecules. Copper (in its different salts, with multiple sites of action, it is applied at a concentration of 1 mL L-1 of water) is allowed days before harvest in conventional and organic production. It is considered low risk with no signs of developing resistance. The other two molecules, azoxystrobin (affects cellular respiration) and fludioxonil (acts on transduction signals, MAP/histidine), in a single mixture, are applied at a concentration of 0.75-1.5 mL L-1 of water in postharvest.
The maximum permissible residual limit is 0.4 ppm in the United States of America, 0.2 ppm in Japan, and 0.1-0.4 ppm in Europe (APEAM, 2016; FRAC, 2022). The objective of the research was to diagnose the causative agent of postharvest anthracnose of apparently healthy avocado fruits, from the Central Pacific Region of Mexico, by morphological characteristics, pathogenicity tests, molecular and phylogenetic identifications, as well as their development under fungicide stress.
In 2019, physiologically ripe avocado fruits, without mechanical damage, visibly healthy and without apparent damage (thrips), were sampled in five orchards in Michoacán, one in Jalisco, and two in Nayarit, Mexico. Twenty fruits per sampling site were stored at 25 °C ±3 °C until symptoms of anthracnose developed (circular brown lesions on the skin that change to darker colors during the ripening process).
Samples of 0.5 cm2 of mesocarp were taken when the first symptoms appeared and were disinfected with 1.5% v/v sodium hypochlorite for 2 min. They were then washed three times with sterile distilled water, dried with sterile blotting cloth, and seeded in Petri dishes with potato dextrose agar medium (PDA; DIBICO, Mexico). They were incubated at 27 °C ±2 °C until mycelium and spores were observed. The culture was isolated by successive reseeding and purified using the monosporic culture technique (Zhang et al., 2013).
For each pathogen isolated, growth rate was measured in PDA medium incubated at 27 °C ±1 °C for 7 days. Every 24 h, the diameter of the colony was evaluated and the growth rate (mm day-1) was calculated. The morphological description was made based on the color of the colony (mycelium), color of conoidal masses, presence of acervuli, size and shape of the spore.
The PCR technique was used to confirm the identity of the isolated strains (Colletotrichum). Genomic DNA was extracted from the mycelium of colonies of 8 d of incubation at 27 °C for each Colletotrichum strain isolated using the CTAB method (Doyle, 1990). The DNA of all isolates was amplified by rDNA-ITS, which included ITS1 (5’CAACTCCCAAACCCCTGTGA-3’) and ITS4 (5’GCGACGATTACCAGTAACGA-3’); and glyceraldehyde-3-phosphate dehydrogenase, where GDF1 (5’-GCCGTCAACGACCCCTTCATTGA-3’) and GDR1 (5’-GGGT GGAG TCGT ACTT GAGC ATGT-3’) were used.
The amplification and cleaning conditions were carried out as indicated by the following authors (Silva-Rojas and Ávila-Quezada, 2011; Fuentes-Aragón et al., 2018; Juárez-Vázquez et al., 2019). The study was conducted at the Comprehensive Phytosanitary Diagnostic Laboratory at the facilities of the College of Postgraduates in the State of Mexico, Mexico.
The evolutionary history was inferred with the Neighbor-Joining method. The Bootstrap consensus tree inferred from 100 replicates was taken to represent the evolutionary history of the analyzed taxa. Branches corresponding to reproductive partitions in less than 50% of collapsed boot replicates. The percentage of replicated trees in which the associated taxa were clustered in the bootstrap test (100 replicates) was shown next to the branches.
Evolutionary distances were calculated using the Tamura-Nei substitution model as tree inference options. The analysis involved 22 nucleotide sequences. All positions containing gaps and missing data were removed. There were a total of 442 positions in the final dataset. Evolutionary analyses were performed in the molecular evolutionary genetics analysis (MEGA) 7.0 software (Weir et al., 2012; Kumar et al., 2016).
Koch’s postulates were used to corroborate the isolated agent as the cause of the observed symptoms. Pathogenicity tests were performed on completely healthy fruits with no visible damage, with a pulp dry matter content between 23-25% (NMX-FF-016-SCFI-2016, 2016). Three wounds (3 mm) were made in the longitudinal area of the fruit and spore suspension (106 spores ml-1) was placed over the wounds. The experiment was repeated twice. All fruits were incubated at 27 °C and 90% relative humidity until anthracnose disease appeared. The development of anthracnose was recorded for each isolate and the pathogen was reisolated to confirm morphological identity and cultural characteristics.
Inhibition of mycelium growth in the presence of two fungicides, copper sulfate and the mixture of fludioxonil + azoxystrobin, was performed in PDA with the concentration of each fungicide; for copper sulfate, they were 20, 40, 60, and 80 ppm; for fludioxonil + azoxystrobin, they were 10, 20, 50, and 70 ppm. PDA alone was included as a control. Five repetitions were performed per C. siamense isolate. A 7-day-old mycelium sample was added to each Petri dish and they were incubated at 27 °C ±1 °C for 48 h (Han et al., 2021). After this incubation period, treatments with fungicides were reinoculated in PDA without fungicide, to verify the fungicidal or fungistatic effect.
All experiments were performed using a completely randomized design with three repetitions, considering the origin of the isolate as a factor of variation for the radial diameter of the colony, the growth rate, the area of the colony at seven days, and the length and diameter of the spore. Mean comparisons were made with the LSD Fisher test (p≤ 0.05).
Nine pathogenic fungal strains belonging to the species complex of C. gloeosporioides were identified. Of these, six pathogens corresponded to the state of Michoacán, in five sampling sites, one pathogenic fungus in Jalisco, and two pathogenic fungi from Nayarit. Fruit disease symptoms, colony characteristics, and spore shape are shown in Figure 1 and described in Table 1. Most of the isolates showed aerial mycelium, white to dark gray color at 7 d of incubation, with orange cone-shaped masses and only in two cases they were black.
All had acervuli. The shape of the spores was cylindrical to ellipsoidal. Growth rate ranged from 11.7 to 12.9 mm day-1, with statistical differences between them (p< 0.05). The length and equatorial diameter of the spore also showed significant differences (p< 0.05). The spores ranged in length from 40.3 to 46.1 μm and from 11 to 15.3 μm in equatorial diameter.
The final diameter of the colony on day seven was different between the strains and ranged from 69 to 83 mm. The concentration of spores at the end of incubation was different between the strains (p< 0.05) (Table 2). Based on these morphological and cultural characteristics, the isolates were identified as C. gloeosporioides sensu lato. Under this classification, different species that cause anthracnose in avocados are grouped.
In pathogenicity tests, typical characteristics of anthracnose disease developed after five days of storage, such as black spots on the pericarp and soft rot on the mesocarp, which spread rapidly throughout the fruit. No other symptoms were observed among the isolates (Figure 2).
Based on the ITS nucleotide sequences and a BLAST search, the isolates obtained in this study were identified as C. gloeosporioides species complex. The nine isolates showed a percentage coverage between 99 and 100% and an identity of 98 to 100% with the genus Colletotrichum and were classified in the gloeosporioides clade based on the ITS nucleotide sequence. With the GAPDH sequence, they showed a percentage of coverage of 79 to 100% with C. siamense, as well as an identity of 98 to 100% with this species (Table 3 and Figures 3 and 4 ).
In all the strains isolated and evaluated, in both fungicides, they achieved 100% inhibition compared to the control (Figure 5). When mycelium samples of the tested pathogens were reseeded in PDA only, the strains developed mycelium within 48 h of incubation. The higher the concentration of the fludioxonil + azoxystrobin fungicide exposed, the lower the mycelium development compared to the control (Figure 6). The Up Cg M4 and Tin Cg M13 strains showed greater susceptibility to low concentrations of the fungicide, compared to the rest of the strains, which, at high concentrations, also developed mycelium, although scarce and disordered.
With the copper sulfate fungicide, all strains developed mycelium after reinoculation in PDA without fungicide. Increased development after exposure to low concentrations of fungicides and decreased development of mycelium after exposure to high concentrations of fungicides. One explanation for this phenomenon of inhibition under stress of both fungicides is that the evaluated strains entered an inactive state in response to the high concentration of the fungicide. This is because, in the field, the concentration of the fungicide decreases over time due to the degradation of the molecule, exposure to ultraviolet light or dilution by rain or environment (Arjona-López et al., 2020; Han et al., 2021).
The identification of C. siamense in the Central Pacific Region of Mexico indicated that the fungus has dispersed within the main avocado-producing regions in Mexico. Research by Weir et al. (2012) mentioned that C. siamense is represented by 30 isolates from a wide range of hosts from various tropical regions and forms a monophyletic clade that cannot be genetically subdivided without being considered a species complex of C. siamense. ITS sequences do not reliably separate C. siamense from the rest of C. gloeosporioides sensu lato (C. alienum, C. fructicola and C. tropicale).
The GAPDH gene sequence was more efficient in distinguishing C. siamense (Lee et al., 2020). Fuentes-Aragón et al. (2020b) reported for the first time in preharvest the presence of C. siamense in fruits with visible symptoms of anthracnose in the Central Pacific Region of Mexico. The results of this research coincided with the studies already mentioned, which indicate that C. siamense is the most abundant pathogen in the Central Pacific Region of Mexico, the one with the highest production and export of ‘Hass’ avocado in Mexico.
The presence of C. siamense, isolated from avocado fruits with visible symptoms of anthracnose in the preharvest stage, was reported in Australia (Giblin and Coates, 2007), New Zealand (Hofer et al., 2021); Mexico (Trinidad-Ángel et al., 2017; Fuentes-Aragón et al., 2020b); Ghana (Honger et al., 2016) and Israel (Sharma et al., 2017). Unlike these studies, this research used avocado fruits without visible symptoms of anthracnose and harvested at physiological maturity, which confirms that C. siamense is an endophytic pathogenic fungus, capable of existing in two conditions of life of the fruit (pre- and postharvest); in addition, it can enter the immature tissue of the fruit (lenticels, pedicels, and direct penetration) and remain dormant for months (dormancy) (Prusky et al., 2013) and further colonize the fruit during storage when the fruit begins to ripen, making it a necrotrophic fungus.
It is evident that the control of anthracnose in preharvest does not eliminate the postharvest problem; therefore, different strategies must be used, one for the control of anthracnose in preharvest and another in postharvest with asymptomatic fruits and thus, keep the quality of the fruits for longer storage, to allow reaching international markets.
On the other hand, in postharvest, due to existing regulations, chemical control is rarely used. Therefore, it is necessary to use alternative methods to the use of chemical compounds to control anthracnose in avocado storage with high efficiency, low cost, and low risk to health and the environment (Herrera-González et al., 2021).
Based on their morphological, molecular and phylogenetic characteristics, they were identified as C. siamense causing anthracnose in postharvest of avocado from the Central Pacific Region of Mexico. In addition, it was evidenced that these strains remain dormant under the stress of the azoxystrobin-fludioxonil and copper sulfate fungicides, but when the stress was removed, growth restarted.
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