In vitro effect of Cuban strains of Trichoderma spp. on the growth of the pathogen Phyllosticta citricarpa (McAlpine) Aa

Citrus black spot (CBS) is caused by the phytopathogenic fungus Phyllosticta citricarpa (McAlpine) van der Aa and affects Citrus spp. This disease is present in regions with summer rainfall climates of Africa, Asia, Australia, and the Americas (11. Farr D, Rossman A. Fungal Databases; Systematic Mycology and Microbiology Laboratory; ARS; USDA. 2024. Retrieved: October 11, 2024 from https://fungi.ars.usda.gov/ ). P. citricarpa causes damages on the appeal of the fruit, but the internal fruit quality remains unaffected. In addition, severe infections may cause premature fruit drop. The presence of CBS is related to production losses, high costs associated with management, and trade restrictions in the fresh produce market. Due to the potential economic impact, P. citricarpa is identified of quarantine importance in CBS-free citrus producing areas (22. Silva-Junior GJ, Feichtenberger E, Spósito MB, Amorim L, Bassanezi RB, et al. Pinta preta dos citros: a doença e seu manejo. Araraquara: Fundecitrus. 2016. Retrieved: October 5, 2024 from http://www.fundecitrus.com.br , 33. Serra W, Manzano León AM. La mancha negra de los cítricos: actualidad y desafíos. Rev. Protección Veg. 2022; 37(3): https://cu-id.com/2247/v37n3e05 ).

Fungicides have been shown to be effective in reducing damage by P. citricarpa and increase yields of quality fruit (22. Silva-Junior GJ, Feichtenberger E, Spósito MB, Amorim L, Bassanezi RB, et al. Pinta preta dos citros: a doença e seu manejo. Araraquara: Fundecitrus. 2016. Retrieved: October 5, 2024 from http://www.fundecitrus.com.br , 33. Serra W, Manzano León AM. La mancha negra de los cítricos: actualidad y desafíos. Rev. Protección Veg. 2022; 37(3): https://cu-id.com/2247/v37n3e05 , 44. Dewdney MM, Walker C, Roberts PD, Peres NA. 2024-2025 Florida Citrus Production Guide: Citrus Black Spot: CPG Ch. 35, CG088/PP279, Rev. 5/2024.EDIS2024 (CPG). Gainesville, FL. 7 pp. https://doi.org/10.32473/edis-cg088-2023 ). Nevertheless, incorrect use of chemicals can lead to selection of resistant fungal strains (55. Souza A. Phyllosticta citricarpa: diversidade genética temporal em pomares de Citrus sinensis e sensibilidade a fungicidas. [PhD Thesis]. Universidad Estatal Paulista Júlio de Mesquita Filho, Facultad de Ciencias Agrarias y Veterinarias. 2015; vii, 74 pp. Retrieved: May 20, 2022 from http://hdl.handle.net/11449/123831 ). In this sense, due to increasing concerns about the environment, human health issues and high costs associated with fungicides, biological-based control strategies are studied as promising alternatives. The control of P. citricarpa with biological agents, such as Trichoderma spp., were examined with encouraging results by treatments with highly suppressive effects on the in vitro pathogen growth (≥ 30%) (66. Kupper KC, Barbosa CE, Moretto C, Bettiol W, Goes A de. Control of Guignardia citricarpa by Bacillus subtilis and Trichoderma spp. Rev. Bras. Frutic. 2011; 33(4): 1111-1118. https://doi.org/10.1590/S010029452011000400009 , 77. Mathelemuse AS, Kena AM. The efficacy of selected biological control agents against citrus black spot (CBS) pathogen Phyllosticta citricarpa. African J. Agricultural Research. 2017; 12: 2101-2104. https://doi.org/10.5897/AJAR2016.11484 ). Trichoderma spp. have been previously reported to be an effective biological control agent of plant pathogens by mechanisms of antibiosis, mycoparasitism, competition for nutrients and space, or induction of host resistance and promotion of plant growth (88. Chien Y, Huang Ch. Biocontrol of bacterial spot on tomato by foliar spray and growth medium application of Bacillus amyloliquefaciens and Trichoderma asperellum. Eur J Plant Pathol. 2020. https://doi.org/10.1007/s10658-020-01947-5 ).

In Cuba, P. citricarpa, which has affected different citrus-productive regions, was first reported in 2007 (33. Serra W, Manzano León AM. La mancha negra de los cítricos: actualidad y desafíos. Rev. Protección Veg. 2022; 37(3): https://cu-id.com/2247/v37n3e05 , 99. Serra W, Lugo Álvarez MB, García Rodríguez D, Alonso-Oliva E, Sanz Llorente A, Guarnaccia V, et al. Polyphasic identification and MAT1-2 isolates of Phyllosticta citricarpa in Cuba. Eur. J. of Plant Pathol. 2022; 162: 995-1003. https://doi.org/10.1007/s10658-021-02453-y ). The management of CBS in Cuba is based on a combination of chemical control and cultural practices. In addition, it should be noted that, although biocontrol agents are part of the management toolbox for production of tobacco (Nicotiana tabacum L.), vegetables, and ornamental plants in our country (1010. Quesada-Mola Y, Fernández-Gonzálves E, Casanueva-Medina K, Ponce-Grijuela E, Márquez-Gutiérrez ME. Actividad biológica de nuevas cepas cubanas de Trichoderma spp. efectivas en el control de Meloidogyne incognita (Kofoid & White) Chitwood. Revista Cubana de Ciencias Biológicas. 2019; 7: 1-9. ISSN: 2307-695X.), this approach have not yet been tested for CBS control. Thus, the objective of this research was to evaluate in vitro of Trichoderma spp. on P. citricarpa growth.

A total of twenty Phyllosticta citricarpa strains (IIFT A1, IIFT A2, IIFT A4 to IIFT A11, IIFT B1 to IIFT B4, IIFT B6, IIFT C1 to C3, IIFT D1, and IIFT D2) were included in this study. They were collected from major Cuban citrus-producing provinces (Matanzas, Cienfuegos, Camagüey and Santiago de Cuba) and identified using a polyphasic taxonomy approach (Table 1) (99. Serra W, Lugo Álvarez MB, García Rodríguez D, Alonso-Oliva E, Sanz Llorente A, Guarnaccia V, et al. Polyphasic identification and MAT1-2 isolates of Phyllosticta citricarpa in Cuba. Eur. J. of Plant Pathol. 2022; 162: 995-1003. https://doi.org/10.1007/s10658-021-02453-y ). The strains of Trichoderma spp., Trichoderma harzianum Rifai (strains LBAT-34 and LBAT-53) and Trichoderma viride Pers. (strain LBAT-TS3), were provided by the Department of Technologies for Biological Media Production at the Plant Health Research Institute (INISAV), located in Havana, Cuba.

Single spore cultures of each fungal isolate were stored on dried filter paper at 4°C and -20°C, as described by Silva-Junior et al. (22. Silva-Junior GJ, Feichtenberger E, Spósito MB, Amorim L, Bassanezi RB, et al. Pinta preta dos citros: a doença e seu manejo. Araraquara: Fundecitrus. 2016. Retrieved: October 5, 2024 from http://www.fundecitrus.com.br ). When needed, small fragments of these colonized filter papers were placed on PDA. Incubation was performed in the dark for 7 days at 27°C. Fungal agar plugs (5 mm diameter), taken from the edge of actively growing mycelium, were used as inoculums for the experiments (99. Serra W, Lugo Álvarez MB, García Rodríguez D, Alonso-Oliva E, Sanz Llorente A, Guarnaccia V, et al. Polyphasic identification and MAT1-2 isolates of Phyllosticta citricarpa in Cuba. Eur. J. of Plant Pathol. 2022; 162: 995-1003. https://doi.org/10.1007/s10658-021-02453-y ).

Growth inhibition of P. citricarpa by Trichoderma spp. was evaluated using the in vitro dual culture assay (77. Mathelemuse AS, Kena AM. The efficacy of selected biological control agents against citrus black spot (CBS) pathogen Phyllosticta citricarpa. African J. Agricultural Research. 2017; 12: 2101-2104. https://doi.org/10.5897/AJAR2016.11484 ). A 5 mm diameter mycelium-agar plug of each P. citricarpa isolate was placed 1 cm from the edge of a fresh PDA plate. After 5 days of incubation (dark, 27°C), plugs of each Trichoderma spp. were transferred to the opposite edge of the plates. Dual cultures were incubated for 7 days and radial growth of P. citricarpa was measured after 3, 5, and 7 days. Percentage of inhibition of radial growth (PIRG) was determined by $\text{PIRG}\left(\text{\%}\right)=\frac{\left(\text{R}1-\text{R}2\right)}{\text{R}1} x 100$ , where R1: radial growth of the pathogen on PDA (control treatment) and R2: radial growth of the pathogen on dual culture (1111. Castro-Albán HA, Castro-Gómez R del P, Alvarado Capo Y. Actividad antifúngica de saponinas de Chenopodium quinoa Willd. frente a hongos fitopatógenos de importancia agrícola. Rev. Protección Veg. 2023; 38: 1-5. https://cu-id.com/2247/v38e22 ). Type and intensity of the antagonism of Trichoderma spp. was classified following Davet et al. (1212. Davet P, Artigues M, Martin C. Production in conditions on aseptiques d' inoculom of Trichoderma harzianum. Ripaipour des essais de lute biological. Agronomic. 1981; 933-936.).

Randomized designs were applied to evaluate the effect of each Trichoderma spp. on P. citricarpa growth. Each experiment with five replicas per treatment was carried out twice. Two colony diameter measurements were taken perpendicular to each other. PIRG data were analyzed by Kruskal-Wallis and non-parametric multiple comparison tests with STATISTICA version 14 software (1313. StatSoft, Inc. STATISTICA (data analysis software system), version 14. 2020, www.stat‐soft.com.).

As results, dual culture tests showed that Trichoderma spp. inhibited the growth of P. citricarpa with significant statistical differences between the PIRGs achieved by the antagonists (H=173.6626; P < 0.001) (Fig. 1).

After 3 days of incubation, the strains of Trichoderma spp. showed PIRG values that ranged from 38.7% to 29.2% (Fig. 1). At this stage of the assay, the highest percentages of radial growth inhibition of P. citricarpa (≥ 35.8%) were obtained with T. harzianum strain LBAT-53 against 12 strains of P. citricarpa (IIFT A1, IIFT A5, IIFT A7, IIFT A8, IIFT A11, IIFT B2 to B4, IIFT C2, IIFT C3, IIFT D1, and IIFT D2); T. harzianum strain LBAT-34 against the IIFT A2 and A7 strains, and T. viride LBAT-TS3 against P. citricarpa strain IIFT B2. The lowest inhibition values (≤ 29.9%) were recorded with T. harzianum strain LBAT-34 against P. citricarpa strain IIFT A4, T. harzianum strain LBAT-53 against strain IIFT A4, and T. viride LBAT-TS3 against strain IIFT B6. As the PIRG values of the remaining treatments did not differ significantly from those mentioned above (Fig. 1), they were considered of intermediate effects.

Interestingly, after 5 and 7 days of co-culture, the antagonist strains overran the P. citricarpa colonies (Fig. 2) and completely inhibited their growth (100% of PIRG). These inhibition values achieved by the strains of Trichoderma spp. did not show statistical differences between them, but they significantly differed from those achieved after 3 days of incubation (Fig. 1).

T. harzianum LBAT-34 and LBAT-53 were previously characterized for the control of the phytopathogenic fungi Rhizoctonia solani J.G. Kühn, Fusarium spp., Agroathelia rolfsii (Sacc). Redhead & Millineux, and Bipolaris oryzae (Breda de Haan) Shoemaker (1414. Pérez EJ, Bernal A, Milanés P, Leiva M, López E, Sierra Y, et al. Influencia del tiempo de incubación de Trichoderma harzianum Rifai en la actividad antifúngica del filtrado de cultivo contra Bipolaris oryzae. Centro Agrícola. 2013; 40(2): 25-30., 1515. Samaniego-Fernández LM, Maimouna OC, Rondón-Castillo AJ, Placeres-Espinosa I. Aislamiento, identificación y evaluación de cepas autóctonas de Trichoderma spp. antagonistas de patógenos del suelo. Rev. Protección Veg. 2018; 33 (3): 1-11. https://opn.to/a/4herH ), while T. viride LBAT-TS3 showed to be antagonistic against nematodes (1616. Fernández E, Casanueva K, Gandarilla H, Márquez ME, Despaigne F, Almandoz J, et al. Nematodos en cultivos protegidos de hortalizas y su manejo en tres localidades de La Habana. Fitosanidad. 2015; 19 (1): 13-22.). The mentioned Trichoderma strains are the basis of the commercially available bio-products TRICOSAVE 34, TRICOSAVE 53, and TRICOSAVE TS3. These products are produced in Cuba to be applied under greenhouse and field conditions as part of an integrated system to control fungi and oomycetes in the production of tobacco, vegetables, and ornamental plants (1010. Quesada-Mola Y, Fernández-Gonzálves E, Casanueva-Medina K, Ponce-Grijuela E, Márquez-Gutiérrez ME. Actividad biológica de nuevas cepas cubanas de Trichoderma spp. efectivas en el control de Meloidogyne incognita (Kofoid & White) Chitwood. Revista Cubana de Ciencias Biológicas. 2019; 7: 1-9. ISSN: 2307-695X.).

Several mechanisms have been reported in the suppression of pathogenic fungi by Trichoderma spp. These include competition for nutrients and space, antibiosis, and hyperparasitism. In the present work, the growth inhibition of P. citricarpa in dual culture prior to physical contact with the antagonist suggested an antibiosis mechanism of Trichoderma spp. This mechanism can be attributed to bioactive compounds of volatile or non-volatile nature produced by the antagonists (1010. Quesada-Mola Y, Fernández-Gonzálves E, Casanueva-Medina K, Ponce-Grijuela E, Márquez-Gutiérrez ME. Actividad biológica de nuevas cepas cubanas de Trichoderma spp. efectivas en el control de Meloidogyne incognita (Kofoid & White) Chitwood. Revista Cubana de Ciencias Biológicas. 2019; 7: 1-9. ISSN: 2307-695X.). Additionally, a hyperparasitic effect was manifested by Trichoderma spp. as observed from the rapid colonization (after 5 days of incubation) and the production of reproductive structures on the pathogen colony. The antagonism of Trichoderma spp. in this study could be classified as of high intensity (++) since the PIRG values were > 25% (1212. Davet P, Artigues M, Martin C. Production in conditions on aseptiques d' inoculom of Trichoderma harzianum. Ripaipour des essais de lute biological. Agronomic. 1981; 933-936.). In this regard, Kupper et al. (66. Kupper KC, Barbosa CE, Moretto C, Bettiol W, Goes A de. Control of Guignardia citricarpa by Bacillus subtilis and Trichoderma spp. Rev. Bras. Frutic. 2011; 33(4): 1111-1118. https://doi.org/10.1590/S010029452011000400009 ) concluded that Trichoderma spp. was a promising antagonist against P. citricarpa, with PIRG values of 31, 37, and 50% after 5 days of co-incubation.

Moreover, the P. citricarpa isolates evaluated in this study included isolates from diverse geographic regions of Cuba, where, also, most of the Cuban citrus is produced. These analyses provide a broader view of the effects of the biological control agents on the P. citricarpa populations present in Cuba.

It should be noted that, although Trichoderma spp. are commonly found in the soil associated with the plant root ecosystem, it can be used successfully for foliar fungal disease management (88. Chien Y, Huang Ch. Biocontrol of bacterial spot on tomato by foliar spray and growth medium application of Bacillus amyloliquefaciens and Trichoderma asperellum. Eur J Plant Pathol. 2020. https://doi.org/10.1007/s10658-020-01947-5 , 1717. Zin NA, Badaluddin NA. Biological functions of Trichoderma spp. for agriculture applications. Annals of Agricultural Sciences. 2020; 65 (2): 168-178. https://doi.org/10.1016/j.aoas.2020.09.003 ). The presence of Trichoderma in the rhizosphere of the plant can induce the systemic resistance mechanism and, therefore, promote the protection of other organs besides the roots (1717. Zin NA, Badaluddin NA. Biological functions of Trichoderma spp. for agriculture applications. Annals of Agricultural Sciences. 2020; 65 (2): 168-178. https://doi.org/10.1016/j.aoas.2020.09.003 , 1818. Yao X, Guo H, Zhang K, Zhao M, Ruan J, Chen J.Trichoderma and its role in biological control of plant fungal and nematode disease. Front Microbiol. 2023; 14:1160551. doi: 10.3389/fmicb.2023.1160551 ). Several formulations of Trichoderma, which may include active spores of this antagonist and/or metabolite extracts, have been shown to be effective for foliar pathogen management. Also, these formulations are recommended for foliar applications or for being directly applied to the soil (88. Chien Y, Huang Ch. Biocontrol of bacterial spot on tomato by foliar spray and growth medium application of Bacillus amyloliquefaciens and Trichoderma asperellum. Eur J Plant Pathol. 2020. https://doi.org/10.1007/s10658-020-01947-5 , 1818. Yao X, Guo H, Zhang K, Zhao M, Ruan J, Chen J.Trichoderma and its role in biological control of plant fungal and nematode disease. Front Microbiol. 2023; 14:1160551. doi: 10.3389/fmicb.2023.1160551 ).

Apparently, this is the first study in Cuba that assesses the in vitro susceptibility of P. citricarpa to biological control agents. Taken together, these results suggest a potential new tool to be included in the management of CBS together with the chemical and cultural strategies currently used. Nevertheless, further evaluations need to be conducted to validate the biocontrol efficacy of Trichoderma against P. citricarpa under production conditions.