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Current agricultural production requires efficient alternatives for pest and disease control that contribute to sustainability within integrated crop management schemes. Additionally, the demands of a globalized market, such as certifications in Good Agricultural Practices like Global G.A.P or Rainforest Alliance, favor the use of environmentally friendly control methods for sustainable production (Sanderson-Bellamy et al., 2016; Figueredo et al., 2018; Nivelo et al., 2020).
One of these alternatives is the use of biopesticides formulated based on antagonistic microorganisms, which can be integrated into crop management strategies, helping to reduce the number of applications and active ingredients of chemical pesticides used in a crop cycle (Samada and Tambunan, 2020). In Colombia, one example is Tricotec® WG developed by the Colombian Corporation for Agricultural Research - AGROSAVIA. Its active ingredient is conidia of the fungus Trichoderma koningiopsis Th003. This biopesticide is registered by the Colombian Agricultural Institute - ICA for use on lettuce (Lactuca sativa), tomato (Solanum lycopersicum), rice (Oryza sativa), blueberry (Vaccinium corymbosum), strawberry (Fragaria vesca), blackberry (Rubus glaucus), potato (Solanum tuberosum) and ornamentals such as rose (Rosa spp.) to control Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani, Sclerotinia sclerotiorum and Sclerotinia minor. It has 50-65% efficacy depending on the production system (Moreno et al., 2020).
However, agricultural production is subject to environmental, anthropogenic, and plant biological conditions which, in addition to yield impacts, can determine pest and disease incidence, sometimes occurring simultaneously.
Therefore, in addition to biopesticides, it is necessary to use chemically synthesized pesticides (whose suspensions or solutions are usually prepared with the addition of adjuvants that facilitate and improve their application and action) since the former can control one or several targets, but do not have a broad spectrum of action (Samada and Tambunan, 2020). For instance, with the applications of Tricotec® WG in rose cultivation, there is control over B. cinerea, but not over Podosphaera pannosa or Peronospora sparsa. For these pathogens, it is necessary to apply fungicides that control them (Debener and Byrne, 2014).
With this in mind, the objective of this work was to determine the effect of adjuvants, fungicides and insecticides used in production systems in which Tricotec® WG is registered for use, on the growth and conidia germination of T. koningiopsis Th003, as well as the effect of the adjuvants on the establishment of the fungus in the phyllosphere (as they are widely used agrochemicals for improving the activity of fungicides and insecticides) with a view to generating recommendations for use according to their potential compatibility with agrochemicals.
Materials and methods
This work was carried out at the Agricultural Microbiology Laboratory of the Tibaitatá Research Center of the Colombian Agricultural Research Corporation - AGROSAVIA.
Microorganism. To evaluate the effect of agrochemicals on the mycelial growth and germination of T. koningiopsis Th003, fungal cultures that had grown for seven and 10 days were used. These cultures were derived from the inoculation of a granule of the biopesticide Tricotec® WG onto potato dextrose agar (PDA) (Oxoid CM0139). Meanwhile, to assess the influence of adjuvants on the fungus’s establishment in the phyllosphere, the commercial form of the biopesticide was utilized.
Agrochemicals evaluated. Twenty-seven fungicides used in crops for which Tricotec® WG is registered for use, 10 insecticides and 7 agricultural adjuvants were evaluated at the highest use dose reported in the technical data sheet of each agrochemical (Tables 1, 2 and 3).
Table 1 Technical information for the fungicides evaluated for their effect on the growth of Trichoderma koningiopsis Th003.
| Fungicida | Grupo químico | Dosis | Mecanismo de acción | Fitopatógeno blanco |
|---|---|---|---|---|
| Mandipropamid | Amidas del ácido mandélico | 1 mL L-1 | Inhibe la síntesis de la pared celular en oomicetes | Phytophthora infestans - Peronospora destructor - Peronospora sparsa -Plasmopara viticola -Peronospora pissi |
| Dimetomorf | Amidas del ácido cinámico | 0.8 g L-1 | ||
| Kasugamicina | Antibiótico aminoglucósido | 1.5 mL L-1 | Inhibe la incorporación de aminoácidos a la síntesis de proteínas | Cercospora apii - Septoria sp. - Venturia inaequalis - Sphaerotheca fuliginea |
| Fluazinam | Fenil piridinaminas | 1 mL L-1 | Afecta la fosforilación oxidativa de las mitocondrias, inhibe la germinación de esporas, formación de apresorios y crecimiento micelial | Botrytis cinerea - Colletotrichum lindemuthianum - Phytophthora infestans |
| Spiroxamina | Spiroketalaminas | 0.6 g L-1 | Inhibe la síntesis de los esteroles | Leveillula taurica - Stemphylium vesicarium - Uncinula necator |
| Dodine | Guanidina | 1.6 mL L-1 | Disolución del estrato lipídico de la membrana llevando a la deshidratación de la célula | Heterosporium echinulatum - Sphaerotheca pannosa - Botrytis cinerea - Mycosphaerella fijiensis - Hemileia vastatrix |
| Iminoctadine Tris (Albesilate) | di-guanidine | 0.75 cml L-1 | Inhibe la síntesis de lípidos, la germinación de esporas, la elongación del tubo germinal y formación del apresorio | Botrytis cinerea - Sphaerotheca pannosa |
| Tiabendazol | Benzimidazoles | 0.9 mL L-1 | Inhibe la división celular a nivel de formación de tubulina | Botrytis cinerea - Cercospora apii - Lasiodiplodia theobromae - Sclerotium cepivorum - Colletotrichum gloeosporioides - Penicillium digitatum |
| Ciazofamida | Cianoimidazoles | 0.2 mL L-1 | Inhibidor de la respiración | Phytophthora infestans A1 - Bremia lactucae - Peronospora farinosa f. sp. spinaciae - Pseudoperonospora cubensis - Peronospora destructor - Pythium ultimum |
| Procloraz | Imidazol | 1.2 mL L-1 | Inhibe la síntesis del ergosterol. | Botrytis cinerea - Colletotrichum gloeosporioides -Colletotrichum lindemuthianum - Alternaria solani - Cladosporium echinulatum |
| Himexazol | Isoxazoles | 2 mL L-1 | Inhibición del crecimiento interfiriendo en la síntesis del ADN y ARN. | Lasiodiplodia theobromae - Gaeumannomyces graminis |
| Flutriafol | Triazol | 0.8 mL L-1 | Alteración de la síntesis del ergosterol, por la inhibición de la demetilación del esteroide | Alternaria porri - Alternaria solani - Sphaerotheca pannosa - Heterosporium echinulatum- Puccinia horiana - Hemileia vastatrix - Helminthosporium oryzae - Sarocladium oryzae - Cercospora oryzae - Pyricularia grisea - Colletotrichum lindemuthianum -Colletotrichum gloeosporioides - Phakopsora pachyrhizi - Mycosphaerella fijensis |
| Hexaconazole | Triazol | 1 mL L-1 | Anti-esporulante, inhibidor de la síntesis de esteroles - ergosterol alterando la estructura y función de la membrana celular | Erysiphe polygoni - Hemileia vastatrix - Puccinia pitteriana - Sphaerotheca pannosa |
| Propiconazole | Triazol | 1.25 mL L-1 | Inhibe la síntesis del ergosterol y esteroles | Helminthosporium sp. - Cercospora oryzae |
| Triadimenol -Tebuconazole | Triazol | 1 mL L-1 | Inhibe la síntesis del esterol y ergosterol | Leveillula taurica - Pyricularia oryzae - Hemileia vastatrix - Puccinia asparagi - Podosphaera leucotricha - Alternaria solani - Lasiodiplodia theobromae - Uncinula necator |
| Tebuconazole -Trifloxystrobin | Triazol - Estrobina | 1 mL L-1 | Inhibe la síntesis de esterol y detiene el transporte de electrones en la cadena respiratoria | Pseudocercospora purpurea - Alternaria solani - Ramularia gossypii - Curvularia spp. - Rhizoctonia solani - Helminthosporium spp - Colletotrichum spp |
| Azoxistrobina-Difenoconazole | Estrobilurina - Triazol | 1.25 mL L-1 | Azoxystrobin inhibe la respiración mitocondrial evitando la formación de energía (ATP), inhibe la germinación de esporas. Difenoconazole inhibe la síntesis del ergosterol. | Alternaria porri - Alternaria solani - Colletotrichum lindemuthianum - Rhizoctonia solani - Helminthosporium oryzae - Colletotrichum gloeosporioides - Botrytis cinerea - Sphaerotheca pannosa |
| Fenhexamid + Tebuconazole | Hidroxianilidas - Triazol | 2 mL L-1 | Inhibe la formación del tubo germinal y síntesis de ergosterol | Botrytis cinerea |
| Propamocarb - Fenamidona | Carbamatos - Imidazol | 2 mL L-1 | Fenamidone inhibe la respiración y formación de esporas. Propamocarb tiene acción antiesporulante e inhibe la síntesis de fosfolípidos y ácidos grasos. | Peronospora viciae - Phytophthora infestans - Peronospora destructor - Pseudoperonospora cubensis - Peronospora sparsa |
| Fenamidone - Fosetyl-Al | Imidazol - Organofosforado | 1 g L-1 | Interfiere en el proceso de respiración celular a nivel de mitocondrias y en la formación de ATP. | Peronospora sparsa |
| Fosetil - Propamocarb | Etil fosfonatos - Carbamatos | 2.5 mL L-1 | Inhibe la germinación, el crecimiento del micelio y esporulación. | Pythium spp - Phytophthora spp - Phytophthora parasitica - Peronospora sparsa |
| Fluopicolide + Propamocarb | Acylpicolides - Carbamatos | 2 mL L-1 | Altera las espectrinas perturbando la estructura celular, afectando la estabilidad del cito esqueleto con la posterior muerte. | Bremia lactucae - Peronospora destructor - Peronospora sparsa - Pseudoeronospora cubensis - Phytophthora capsici - Phytophthora infestans - Phytophthora palmivora |
| Pyrimethanil -Iprodione | Pirimidina - Dicarboximidas | 1 mL L-1 | Pyrimethanil inhibe la secreción de enzimas. Iprodione inhibe la germinación, elongación del tubo germinal, producción de conidios y crecimiento micelial | Botrytis cinerea |
| Fluopyram + Pyrimethanil | Piridiniletilbenzamidas - Pirimidina | 1 mL L-1 | Inhibidor del succinato deshidrogenasa actuando en la cadena respiratoria e inhibe la secreción de enzimas. | Botrytis cinerea - Cladosporium spp. - Mycosphaerella fijiensis |
| Fludioxonil + Ciprodinil | Fenilpirroles - Pirimidina | 0.6 g L-1 | Ciprodinil afecta la síntesis de metionina y la secreción de enzimas hidrolíticas. Fludioxonil inhibe la proteína kinasa en la ruta de transducción de la señal de osmosensibilidad | Botrytis cinerea |
| Thiram + Pyrimethanil | Ditiocarbamato - Pirimidina | 2 mL L-1 | Inhibe la secreción de enzimas hidrolíticas, la germinación de esporas y el crecimiento micelial | Botrytis cinerea |
| Metalaxil-M - Mancozeb | Anilida - Ditiocarbamatos | 3 g L-1 | Inhibe la respiración y la germinación de las esporas | Plasmopara viticola - Peronospora sparsa - Phytophthora infestans |
The information contained in this table comes from the technical data sheets of the suppliers of the commercial products.
Table 2 Technical information for the insecticides evaluated for their effect on the growth of Trichoderma koningiopsis Th003.
| Insecticida | Grupo químico | Dosis | Mecanismo de acción | Patógeno blanco |
|---|---|---|---|---|
| Spinosad | Naturalite | 0.5 mL L-1 | Actúa por contacto e ingestión, causando parálisis. Activa el receptor de la acetilcolina nicotínica, en diferente sitio que la nicotina o imidacloprid. | Frankliniella occidentalis - Liriomyza spp. - Thrips palmi - Tuta absoluta - Spodoptera frugiperda |
| Dinotefuran | Neonicotinoides | 0.5 mL L-1 | Interfiere la neurotransmisión a través de los receptores nicotínicos. | Frankliniella occidentalis - Oebalus insularis |
| Sulfoxaflor | Sulfoxaminas | 1 mL L-1 | Neurotóxico trabaja en el sistema nervioso central en los receptores nicotínicos de acetilcolina generado excitación generalizada, parálisis, postración y finalmente la muerte de las plagas. | Collaria sp. - Trialeurodes vaporariorum - Trialeurodes vaporariorum - Empoasca kraemer - Aphis gossypii -Bemisia tabaci - Oebalus poecilus - Diaphorina citri Kuwayama |
| Spiromesifen | Derivados del ácido tetrónico (ketoenoles) | 0.5 mL L-1 | Interfiere con la síntesis de lípidos, afectando el desarrollo y fecundidad. | Oligonychus yothersi - Trialeurodes vaporariorum - Bemisia tabaci - Tetranychus urticae - |
| Malathion | Organofosforados | 1mL L-1 | Inhibidor de la acetilcolinesterasa | Frankliniella occidentalis - Anthonomus grandis - Thrips tabaci - Trialeurodes vaporariorum - Collaria scenica |
| Beta-cyfluthrin + Imidacloprid | Neonicotinoide - Piretroide | 1 mL L-1 | Se une en forma postsináptica a los receptores nicotinérgicos y afecta el canal del sodio, en el sistema nervioso. | Premnotrypes vorax - Trialeurodes vaporariorum -Anthonomus grandis - Eutheola bidentata - Spodoptera frugiperda - Oebalus poecilus - Thrips tabaci - Thrips palmi - Gryllotalpa hexadactyla - Frankliniella occidentalis - Diaphorina citri |
| Chlorfenapir | Pirroles | 0.6 mL L-1 | Interrumpe la generación de energía por la liberación y extracción de protones H+ antes de llegar al ATP. | Frankliniella occidentalis - Tetranychus urticae - Tuta absoluta (Meyrick) |
| Metoxifenocide | Benzoilhidracina | 1 mL L-1 | Mimético de la hormona de la muda (ecdisona) que acelera el proceso de muda y con acción ovicida | Anticarsia gemmatalis - Rachiplusia nu - Helicoverpa gelotopoeon - Spodoptera frugiperda - Heliothis virescens - Cydia pomonella - Tuta absoluta -Lobesia botrana |
| Piriproxifen | Fenil éter | 0.6 mL L-1 | Interferencia de la hormona juvenil “HJ” causando la inhibición de metamorfosis, de embriogénesis, de la reproducción, del desarrollo larvario y perturbación de la diapausa | Bemisia tabaci - Trialeurodes vaporariorum - Liriomyza sp. - Frankliniella occidentalis -Thrips tabaci - Heliothrips haemorrhoidalis - Bombacoccus aguacatae - Hemiberlesia lataniae - Aspidiotus nerii - Aleuropleurocelus spp. |
| Permetrina | Piretroide | 0.6 mL L-1 | Interrumpe la función de las neuronas por interacción con los canales de sodio. | Spodoptera frugiperda - Epitrix sp. - Tecia solanivora - Plutella xylostella - Tuta absoluta |
The information contained in this table comes from the technical data sheets of the suppliers of the commercial products.
Table 3 Technical information for the adjuvants evaluated for their effect on the growth of Trichoderma koningiopsis Th003.
| Nombre comercial | Principio activo | Dosis |
|---|---|---|
| Agrotin® SL | Polisacáridos, alcoholes polivinílicos, siliconas | 1 mL L-1 |
| HIPOTENSOR SYS | Polietilenglicol - Polidimetilsiloxano, fosfatos mono y dipotásicos | 1 mL L-1 |
| MF REDUX® | Alquil alcohol poliglicol éter | 1 mL L-1 |
| Fluyex® | Alcohol etoxilado modificado | 5 mL L-1 |
| INEX-A® | Alquil Polieter Alcohol Etoxilado, Alquil Poliglicol, Aril Polietoxietanol | 4 mL L-1 |
| Carrier® | Ácidos carboxílicos insaturados y glicéridos saturados | 1.5 mL L-1 |
| Bioplant® | Aril polietoxietanol - Poliglucósido etoxilado - | 1 mL L-1 |
The information contained in this table comes from the technical data sheets of the suppliers of the commercial products.
Effect of agrochemicals on mycelial growth of T. koningiopsis Th003. Petri dishes were prepared with Dextrose Sabouraud agar (Scharlau 01-165-500) supplemented with each agrochemical, and the same agar without agrochemicals was used as a control. A 5 mm disk of T. koningiopsis Th003 mycelium taken from the fungal culture on PDA was placed in the center of each dish. The Petri dishes were incubated for 120 hours at 25 °C and at the end of this time the diameter of the colonies was read. With the data obtained, the percentage of inhibition was calculated using the formula: Inhibition (%) = ((X - Y) / X) x 100, where X = is the diameter of the colony of T. koningiopsis Th003 in the control dishes, Y = the diameter of the colony of T. koningiopsis Th003 in the dishes supplemented with each of the agrochemicals.
Effect of agrochemicals on the germination of T. koningiopsis Th003 conidia. The conidia were collected from a fungal culture on PDA, making a suspension that was adjusted to a concentration of 1x107 conidia mL-1 using the Neubauer chamber counting technique. Subsequently, 1 mL was taken and transferred to Erlenmeyers flasks containing 20 mL of the solutions of each agrochemical according to the established doses (Tables 1, 2 and 3). The fungal conidia suspended in water were used as a control. The inoculated solutions were left to stand for one hour at 17 °C, simulating the time they could remain in a fumigation equipment during application. Subsequently, 100 μL were taken from each Erlenmeyer and placed in Petri dishes with water agar, spreading them over their surface with a Drigalsky rake. The dishes were incubated for 24 hours at 25 °C and after this, the number of germinated and non-germinated conidia was read by counting 100 conidia on a 1 cm2 agar square, taking three squares from each dish. The germination percentage was determined using the formula: Germination (%) = (Germinated conidia / Total conidia) x 100. A spore was considered as a germinated spore when the length of the germ tube was at least 50% of the length of the non-germinated spore (Muy-Rangel et al., 2018).
Effect of adjuvants on the establishment of T. koningiopsis Th003 in the phyllosphere. Suspensions of Tricotec® WG (at a concentration of 1x106 conidia mL-1) were prepared in solutions of each adjuvant, left to stand for one hour, and then applied to five-month-old cape gooseberry (Physalis peruviana) plants in a field crop. The biopesticide prepared in water at the concentration described above was applied as a control. Seven days later, 10 leaves were taken from the plants and packed in paper bags for analysis in the laboratory. The leaves were cut into 1 cm2 fragments using a sterile stainless steel punch, 10 g were taken and placed in Erlenmeyers with 90 mL of 0.1% Tween 80 (stock suspension) and left in constant agitation at 150 rpm for one hour. Subsequently, a 1:10 dilution was made from each, which was vortexed for 30 seconds. From each stock suspension and dilution, 100 μL were taken and placed in Petri dishes with Rose Bengal + chloramphenicol agar (Oxoid CM0549) (three dishes per dilution), the aliquot was distributed homogeneously with a Drigalsky rake. The dishes were incubated at 25 °C for 5 days, after which colony forming units (CFU) were counted and the results expressed as Log (CFU g-1).
Experimental design and data analysis. The assays were established under a completely randomized experimental design. The experimental unit (EU) corresponded to a Petri dish, with 10 replicates for the evaluation of the effect of agrochemicals on mycelial growth and three replicates for the evaluation of the effect on conidial germination. Data were subjected to analysis of variance and means were compared by Fisher’s LSD test (*= P>0.05) using Statistix 10.0 statistical software.
Results
Effect of agrochemicals on mycelial growth of T. koningiopsis Th003. In experiments testing the impact of fungicides on mycelial growth, no inhibition was observed in the presence of Cyazofamid, Dimethomorph, Fenamidone - Fosetyl-Al, Fosetyl - Propamocarb, Fluopicolide - Propamocarb, Hexaconazole, Mandipropamid, and Propamocarb - Fenamidone. There were no significant differences between these fungicides and the control (Figure 1).
Figure 1 Inhibition of mycelial growth of T. koningiopsis Th003 on Sabouraud agar supplemented with fungicides. Columns with the same letter are not significantly different according to Fisher LSD test (*= P>0.05)
However, when the fungus encountered a medium containing Flutriafol, Kasugamycin, and Fenhexamid - Tebuconazole, there was a growth inhibition ranging from 34 to 48%. In the presence of Azoxystrobin - Difenoconazole, Dodine, Fluazinam, Fludioxonil - Cyprodinil, Pyrimethanil - Iprodione, Iminoctadine Tris (Albesilate), Himexazole, Propiconazole, and Tebuconazole - Trifloxystrobin, the inhibition ranged from 60 to 79% (Figure 1). While Thiram - Pyrimethanil, Prochloraz, Thiabendazole, Spiroxamine, and Triadimenol -Tebuconazole completely halted the growth of T. koningiopsis Th003 (Figure 1).
In relation to insecticides, only Beta-Cyfluthrin - Imidacloprid Permethrin and Malathion reduced fungal growth, inhibiting it by 23% and 44% respectively. These were also the sole insecticides to exhibit significant differences compared to the control (Figure 2).
Figure 2 Inhibition of mycelial growth of T. koningiopsis Th003 on Sabouraud agar supplemented with insecticides. Columns with the same letter are not significantly different according to Fisher LSD test (*= P>0.05).
Regarding the adjuvants, Carrier® had no effect on the mycelial growth of T. koningiopsis Th003. However, other adjuvants inhibited growth between 44 and 70%, with Fluyex® showing the maximum inhibition (Figure 3).
Figure 3 Inhibition of mycelial growth of T. koningiopsis Th003 on Sabouraud agar supplemented with adjuvants. Columns with the same letter are not significantly different according to Fisher LSD test (*= P>0.05).
Effect of agrochemicals on the germination of conidia of T. koningiopsis Th003. In the presence of Dodine, Metalaxyl-M - Mancozeb and Thiram - Pyrimethanil, conidial germination was totally inhibited, while Iminoctadine Tris (Albesilate) only allowed it in 14% and Fluazinam and Plocloraz in 63% (Figure 4). Meanwhile, in the presence of the other fungicides the germination of conidia was ≥ 87%, 13 of them without presenting significant differences with respect to the control.
Figure 4 Germination of T. koningiopsis Th003 conidia exposed to fungicide solutions. Columns with the same letter are not significantly different according to Fisher LSD test (*= P>0.05).
As for the insecticides, the conidia in the presence of Chlorfenapyr presented a germination of 84%, lower than that obtained with the other insecticides, where a germination of ≥ 97% was obtained, without presenting significant differences with respect to the control (Figure 5). With respect to the adjuvants, five of them presented significant differences with respect to the control; however, germination in the presence of all of them was ≥ 93% (Figure 6).
Figure 5 Germination of conidia of T. koningiopsis Th003 exposed to insecticide solutions. Columns with the same letter are not significantly different according to Fisher LSD test (*= P>0.05).
Figure 6 Germination of conidia of T. koningiopsis Th003 exposed to adjuvant solutions. Columns with the same letter are not significantly different according to Fisher LSD test (*= P>0.05).
Effect of adjuvants on the establishment of T. koningiopsis Th003 in the phyllosphere. The highest populations of T. koningiopsis Th003 were recovered in the treatments corresponding to Agrotin® SL and HIPOTENSOR, with means of 3.5 and 3.3 log (CFU g-1) respectively, showing significant differences compared to the other treatments. The control presented a mean of 2.9 log (CFU g-1) and together with Fluyex® and INEX-A®, with means of 2.7 log (CFU g-1), showed the lowest values for recovered populations of the fungus (Figure 7).
Discussion
The fungicides used for controlling oomycete phytopathogens (Cyazofamid, Dimethomorph, Fenamidone - Fosetyl-Al, Fosetyl - Propamocarb, Fluopicolide - Propamocarb, Hexaconazole, Mandipropamid, Propamocarb - Fenamidone) did not affect mycelial growth or conidial germination of T. koningiopsis Th003 (with the exception of Metalaxyl-M - Mancozeb). Oomycetes have a cell wall consisting of cellulose and sitosterol as membrane lipid (Restrepo et al., 2016), unlike fungi which have chitin and ergosterol (Gow et al., 2017). Since some of these fungicides negatively affect the oomycete cell wall, the lack of cellulose and sitosterol in T. koningiopsis Th003 may explain why they did not inhibit growth.
Meanwhile, other fungicides used against B. cinerea, Colletotrichum sp., Alternaria sp., Helminthosporium sp, or Rhizoctonia solani, inhibited mycelial growth up to 100%, indicating toxic effects. However, only Dodine, Metalaxyl-M - Mancozeb, Thiram - Pyrimethanil and Iminoctadine Tris (Albesilate) affected conidial germination, while with others, germination was less impacted, perhaps due to tolerance as reported for some Trichoderma species against certain fungicides (Escudero-Leyva et al., 2022).
Agrochemicals used as adjuvants and insecticides can stimulate or inhibit mycelial growth as well as the germination of conidia of antagonistic fungi (Rashid et al., 2012; Sain et al., 2022). These effects depend on the chemical properties of the agrochemical, the concentration used, and the biology of the fungus. For example, a study by Sabogal-Vargas et al. (2023) showed that the insecticide chlorpyrifos at concentrations of 960, 1,200 and 1,440 mg L-1 increasingly inhibited mycelial growth of Trichoderma asperellum TCA3, T. asperellum TCA21 and T. harzianum TCA23. However, germination of T. asperellum TCA21 conidia was not inhibited. The inhibition of conidia germination in the other strains was attributed to an accumulation of self-inhibitors of germination, causing a state of dormancy (Sabogal-Vargas et al., 2023).
For some insecticides and adjuvants, the inhibitory effect on mycelial growth but not conidial germination has been related to alteration of the electrostatic charge of the fungal surface and potential elimination of the mucous layer covering the conidia. This may occur by interruption of metabolism in the cell wall and its effect on membrane permeability (Clifford and Hislop, 1975; Rashid et al., 2012; Fait et al., 2019). The active ingredient of the biopesticide is conidia of the fungus. When applied in the phyllosphere suspended in adjuvant solutions, the conidia germinated and the fungus became established despite the presence of adjuvants and insecticides. Consequently, populations of T. koningiopsis Th003 recovered were higher or similar to the control. Thus, both the adjuvants and insecticides evaluated could potentially be applied together with the biopesticide, a condition that also applies to the fungicides that did not affect conidial germination.
The tolerance and resistance of a fungus to fungicides is considered an evolutionary process, where exposure to an active ingredient exerts selection pressure on a population, killing the initial wild population but not the altered mutant population. Thus, overexposure accelerates changes towards tolerant and resistant populations, which have developed mechanisms such as alteration or overexpression of the fungicide target site, detoxification, and exclusion or expulsion from the site of action (FRAC, 2019).
However, the tolerance that T. koningiopsis Th003 showed for some agrochemicals, particularly fungicides, may be innate to the fungus’s metabolism. In the production of the Tricotec® WG biopesticide, the fungus used is not exposed to any agrochemicals, as it comes from a germplasm bank rather than being recovered from the environment. This implies that T. koningiopsis Th003 may possess inherent stress tolerance genes, as reported for other Trichoderma species. It may also produce degradative enzymes or proteins responsible for regulating agrochemical degradation processes, allowing it to tolerate and grow in their presence (Tripathi et al., 2013; Ramangouda et al., 2023).
Conclusions
The fungicides used to control oomycetes (Cyazofamid, Dimethomorph, Fenamidone - Fosetyl-Al, Fosetyl - Propamocarb, Fluopicolide - Propamocarb, Hexaconazole, Mandipropamid, Propamocarb - Fenamidone) did not affect mycelial growth or conidia germination of T. koningiopsis Th003. In contrast, those used against ascomycetes or basidiomycetes showed some inhibition. Mycelial growth was completely inhibited by Thiram - Pyrimethanil, Prochloraz, Thiabendazole, Spiroxamine and Triadimenol -Tebuconazole. Thiram - Pyrimethanil, Dodine and Iminoctadine Tris (Albesilate) also inhibited conidial germination. The insecticides Beta-Cyfluthrin - Imidacloprid, Permethrin and Malathion inhibited mycelial growth but did not affect conidia germination. Most adjuvants inhibited mycelial growth by 44-70% but did not impact conidia germination or fungal establishment on the leaf surface. Carrier® was the exception with no effect. Thus, considering integrated pest management strategies, it is possible to mix adjuvants, insecticides and fungicides that did not inhibit T. koningiopsis Th003 conidia germination with the biopesticide.
