elocation-id: e3392
Coffee rust (Hemileia vastatrix) is one of the most economically important diseases in coffee crops due to the production losses of up to 50% that it causes. In addition to the conventional chemical control of this disease, several natural enemies, such as mycoparasitic fungi, regulate its incidence and severity. The study aimed to identify and characterize five strains of fungi associated with the natural control of H. vastatrix isolated from the coffee-growing area of Turrialba, Cartago, Costa Rica. Five strains of Simplicillium were identified by molecular analysis; measurements of macroscopic characteristics, microscopic characteristics, radial growth, and conidia production were made. According to the analysis in the NCBI database, the five strains matched the species Simplicillium lanosoniveum. Overall, macroscopic characteristics were similar between the isolates; the most notable difference was the coloration of the culture medium. Strains that showed a color change from white to brown had lower radial growth. The arrangement of the phialides, the shape and size of the conidia, and the microscopic features were generally consistent with the genus Simplicillium. Conidia size and production were statistically different between strains (p˂ 0.0001). In addition, the strains with the largest conidia recorded the lowest values of conidia production. These results provide the first report that characterizes strains of the genus Simplicillium associated with H. vastatrix in Costa Rica.
cultural characterization, natural biological control, physiological characterization.
Coffee crops face multiple diseases, with coffee rust (Hemileia vastatrix) being one of the most economically significant. This disease causes premature leaf to drop and branch drying, which can result in a loss of more than 50% of the crop and even the death of plants (Aristizábal and Johnson, 2022).
The most devastating rust outbreak in Central America occurred during the 2012-2013 cycle, with estimated losses exceeding 499 million dollars, leading several countries to declare a national emergency (Promecafé, 2013; IICA, 2013). The usual control methods for this disease include the exclusive use of conventional fungicides or copper-based protectors; nevertheless, these have effects that can affect the microbial composition of the soil, altering the functions and biochemical processes that occur in it, which also caused plants to be more susceptible to diseases (Meena et al., 2020). Alternatively, sustainable management techniques can be implemented through the use of natural enemies of H. vastatrix associated with coffee rust (García-Nevárez et al., 2019).
Previously, the mycoparasitic fungus Lecanicillium lecanii (Zimmerman) (Guharay et al., 2001) was reported as a biological controller of H. vastatrix (Santiago-Elena et al., 2020). This mycoparasite attacks when rust is active (within the living tissue of the host) by introducing its germ tube into cell membranes to later produce metabolites that inhibit germination, affect the morphology of uredospores, and cause the death of the fungus (Moricca and Ragazzi, 2008).
Due to the above, when isolating parasitic fungi from rust, it is assumed that the isolated fungus is Lecanicillium (Verticillium), without further evidence to support its identification. This assumption can be risky, especially when new research protocols are initiated. The inclusion of new isolated fungi associated with the natural control of pests or diseases requires correct identification before establishing experiments focused on a biological control program and it is essential for the production of quality biopesticides (Safavi, 2010).
This process must be carried out by specialized taxonomists by identifying morphological characteristics, such as the shape, size, and germination speed of the conidia, as well as the appearance of the colony (shape, elevation, surface appearance, and color) (Hajek and Leger, 1994) and requires the use of molecular markers for their proper identification. Molecular identification by amplification of the Internal Transcribed Spacer (ITS) offers fast and reliable results for most fungal taxa, which is why it is currently considered one of the main diagnostic tools (Lücking et al., 2020).
The study aimed to perform a cultural and physiological characterization and a molecular identification of fungal strains associated with H. vastatrix pustules in the coffee-growing area of Turrialba, Costa Rica.
The study included five fungal strains from the coffee-growing area of Turrialba, Cartago, Costa Rica in the localities of San Juan Norte (SJ), Jabillos (JV), Aquiáres (AQ), Santa Rosa (SR), and CATIE (EC), where the coffee trees were located between 600 and 950 masl. The samples were coffee leaves with advanced lesions of rust and fungi growing on them (Figure 1).
Strain isolation. It was performed by seeding the inoculum in plates with potato dextrose agar (PDA) medium; this inoculum was obtained from the pustules that presented a cottony growth on them.
Macroscopic characterization. Monosporic isolates of 10 colonies per strain were performed by dilutions (10-4, 10-5 and 10-6); these were seeded and incubated at 25-28 °C for 15 days and then the color (Munsell Color Company 1988), the shape, elevation and striation formed were recorded according to the criteria proposed by Fox (1999).
The genetic material was extracted according to the protocol described by Kuske et al. (1998), followed by an amplification of the ITS region using the primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) by polymerase chain reaction (PCR); the product obtained from the amplification was visualized in a 1% agarose gel for subsequent sequencing. The consensus of the sequences was carried out with the BioEdit program and they were compared with those reported in the National Center for Biotechnology Information (NCBI) database using the Blast tool.
The sample size necessary to measure the size of the conidia was determined by measuring groups with different numbers of conidia and calculating the standard deviation (SD) in each case. The sample size that would guarantee a stable SD was selected (Cortez et al., 2003). Based on these results, measurements were made in five replications of monosporic colonies of each strain. In each replication, four groups of conidia were measured, observing 35 conidia per group, using an Omax ToupView® x 86 camera mounted on a pre-calibrated compound microscope.
The serial dilution technique was used to isolate each monosporic strain and culture them in PDA in Petri dishes. After 10 days of incubation at 25-28 °C, the average radial diameter was measured with a Truper® Caldi-6 MP digital vernier. To assess conidia production, one cm2 of each strain was suspended in 10 mL of sterile distilled water with 0.1% Tween 80%, subjected to an ultrasonic bath for three minutes, and stirred in a vortex for one minute. The conidia were counted with a Neubauer chamber. Radial growth was measured and conidia were counted in 10 colonies per strain.
The experiment was conducted using a completely randomized design with 10 replications of each colony to assess radial growth and five to assess conidia size. The analysis was performed using general linear and mixed models with the InfoStat statistical package. For the variables of number and size of conidia, the DGC mean separation test (p= 0.05) was used in order to eliminate the lack of the transitive characteristic between means and facilitate the discussion of the results. For the radial growth variable, the LSD test was performed (p≤ 0.05).
Macroscopic characterization. The most noticeable difference between strains was the color change at the bottom of the Petri dish. The JV and SR strains exhibited an olive yellow coloration, whereas EC, AQ and SJ presented yellow, white, and pale yellow coloration, respectively. The white coloration of the mycelium was consistent across all strains, as were the circular shape and raised edge. Only AQ showed abundant striae in the culture medium during growth (Table 1), which could sometimes be seen from the top of the colony (Figure 2).
The size of the conidia was significantly different between strains (p≤ 0.003). The EC, AQ, and JV strains, which had a size of 1.97, 1.94 and 1.88 μm, respectively, were statistically the same (DGC= 0.05), but with a significant difference compared to SR (1.75 μm) and SJ (1.73 μm) (Figure 3).
Microscopic characterization. The division of the genus Verticillium by Zare and Gams (2004) is based on systematic studies, separating taxa formed by solitary phialides from the genus Lecanicillium and placing them within the genus Simplicillium. From this perspective, the microscopic characteristics of the evaluated strains coincide with those of the latter genus. Oval conidia (1.7 to 1.97 μm) were also observed within globose heads that were suspended from solitary and sometimes prostrate phialides (Figure 4).
Radial growth and conidia production. There was a highly significant difference between the strains with respect to radial growth after five and ten days of growth in PDA (p˂ 0.0001). The strains that showed the highest growth in the five days were SJ and EC with 18.9 and 18.39 mm, respectively, whereas SR, AQ and JV showed the lowest values with 16.44, 15.67 and 15.65 mm, respectively, and did not differ statistically from each other (p≥ 0.05). After 10 days, the highest growth values were obtained by the SJ (36.1 mm) and EC (33.5 mm) strains, followed by AQ (28.8 mm), whereas SR (26.8 mm) and JV (25.7 mm) showed the lowest values. These results indicate a high variability within the same genus (Figure 5).
Conidia production varied significantly between strains (p˂ 0.0001). The SJ, JV, and SR strains showed higher production with values of 1.4×107, 1.3×107 and 1.2×107 cm-2, whereas the SR and SJ strains presented the smallest conidia size (1.75 and 1.73 μm). On the contrary, it was recorded that the EC and AQ strains had larger structures (1.97 and 1.94 μm, respectively); however, they produced significantly less; EC produced 6.8×106 conidia cm-2 and AQ 4.4×106 conidia cm-2, values that did not differ statistically from each other (p≥ 0.05) (Figure 6).
Molecular identification. The EC, AQ, SR, SJ, and JV strains isolated and analyzed in the NCBI database matched the species Simplicillium lanosoniveum. The sequences for each identified strain were selected to be registered in the NCBI GenBank database (Table 2).
The results of this study contrast with other publications involving the natural biological control (NBC) of H. vastatrix in the same region of Costa Rica. This difference is mainly due to previous publications pointing to the fungus L. lecanii as the main natural enemy of coffee rust (Vandermeer et al., 2009; Jackson et al., 2012); in contrast, in the present research, the strains analyzed corresponded to Simplicillium lanosoniveum. The incorrect determination of the natural controller of H. vastatrix may be caused by the similar appearance of the fungus while parasitizing rust; both Simplicillium and Lecanicillium form a white cottony coating. In addition, these genera share morphological characteristics, which often leads to incorrect classification (Lim et al., 2014).
The cultural characteristic with the greatest variation between strains was the color of the colonies seen from the bottom of the Petri dish. One explanation for this could be the formation of resistance structures submerged in the culture medium; an aspect described by Inderbitzin et al. (2011) in Verticillium species. After 15 days of growth, it was observed that the JV and SR strains tend to form resistance structures in less time since the color of the submerged mycelium changed from white to olive yellow (2.5Y 6/6 on the Munsell scale) during incubation; similar observations were recorded by Cortez et al. (2003) on Lecanicillium isolates.
The tendency of the strains to create resistance structures in a shorter time may be a consequence of lower radial growth, as observed in our results that show that the JV and SR strains presented the lowest growth values (Figure 5). In addition, color changes in the culture medium may be associated with the strain’s ability to produce secondary metabolites (VanderMolen et al., 2013).
Since information on the ability of Simplicillium to produce conidia is very limited at this time, comparisons can be made with related taxa such as Verticillium or Lecanicillium. James et al. (2016) found a high diversity of fungi by molecular analysis in coffee leaf discs infected by rust. Based on comparisons of leaf discs without the presence of rust, the authors assumed that there were 15 possible mycoparasitic fungi.
Nonetheless, no effectiveness tests were conducted to examine whether these fungi actually had any degree of control over H. vastatrix. In a study conducted in coffee-growing areas of Veracruz, Mexico, Gómez et al. (2017) isolated mycoparasitic fungi from rust pustules and found a fungus of the genus Simplicillium with high biological control properties. In a recent study by Si et al. (2022) they recorded S. lanosoniveum as a new hyperparasite of the wheat rust Puccinia graminis, despite being a different crop, this research also confirms that this fungus is associated with the biological control of rusts.
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