LUCAS GUEDES SILVA DESENVOLVIMENTO DE FORMULAÇÕES DE Trichoderma PARA USO NA PROMOÇÃO DE CRESCIMENTO DE PLANTAS E CONTROLE DE Sclerotinia sclerotiorum Botucatu 2022 LUCAS GUEDES SILVA DESENVOLVIMENTO DE FORMULAÇÕES DE Trichoderma PARA USO NA PROMOÇÃO DE CRESCIMENTO DE PLANTAS E CONTROLE DE Sclerotinia sclerotiorum Tese apresentada à Faculdade de Ciências Agronômicas da Unesp Câmpus de Botucatu, para obtenção do título de Doutor em Agronomia/Proteção de Plantas. Orientador: Dr. Wagner Bettiol Coorientadora: Cristiane Sanchez Farinas Botucatu 2022 AGRADECIMENTOS Agradeço primeiramente a Deus, por ter me guiado em todos os momentos para que pudesse concluir esta grandiosa etapa de minha vida. À Universidade Estadual Paulista “Júlio de Mesquita Filho”/Faculdade de Ciências Agronômicas, pela estrutura e oportunidade, professores e funcionários, pelos ensinamentos e exemplos transmitidos. À Embrapa Meio Ambiente e à Embrapa Instrumentação, pela estrutura, oportunidade e apoio concedidos para meu aperfeiçoamento pessoal e profissional. Ao Prof. Dr. Wagner Bettiol, pelos ensinamentos, dedicação, orientação, apoio, confiança e incentivo. Ao Dr. Gabriel Moura Mascarin, pelos ensinamentos, parceria, apoio e colaboração no trabalho. À banca examinadora, pela disponibilidade e valiosas contribuições para melhoria deste trabalho. Agradeço imensamente aos meus pais, Jaime e Sônia, pelo amor, apoio incondicional, ensinamentos e por entenderem as minhas ausências. À minha irmã Karen, pela amizade e companheirismo. À minha namorada Rafaela, pelo carinho, cuidado, incentivo e por ter me acompanhado nesta etapa. Agradeço aos colegas de trabalho do Laboratório de Microbiologia Ambiental “Raquel Ghini” da Embrapa Meio Ambiente, pela amizade, companheirismo e por vivenciar momentos tão importantes comigo. Aos meus amigos da Republica Zona Azul e do Apartamento 43, onde passei grandes momentos: Tiago, Dennis, Alberto, Lucas, Vitoldo, Vinicius, Murilo, Rodrigo, Diego, Laudelino, Ricardo, Caetano, Diego, Bárbara, Peterson, João, Davi e Carlos. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil – CAPES – Código de financiamento 001. Meu muito obrigado a todos, que de alguma forma se fizeram presentes e vibram comigo por esta tão esperada conquista! RESUMO Fungos do gênero Trichoderma apresentam um complexo arsenal de mecanismos envolvidos na proteção de plantas, os quais incluem supressão de fitopatógenos, promoção de crescimento e mitigação de estresses abióticos em plantas. Para tanto, a seleção dos isolados é de fundamental importância, pois Trichoderma spp. são altamente diversificadas em eficácia na supressão de patógenos de plantas, apresentando respostas variadas de acordo com as cepas que estão sendo confrontadas. Outros desafios são relacionados à multiplicação, ao armazenamento, e ao desenvolvimento de formulações estáveis e com vida de prateleira adequada. Vencer esses desafios colaborará para a disponibilização de produtos com qualidade adequada no mercado. O presente trabalho teve como objetivos selecionar isolados de Trichoderma spp. promotores de crescimento em algodoeiro e inibidores da germinação de escleródios de Sclerotinia sclerotiorum; otimizar a produção de Trichoderma asperelloides em farinha de arroz e; desenvolver formulações granulares à base de farinha de arroz. Trichoderma asperelloides CMAA 1584 apresentou maior eficiência no controle de Sclerotinia sclerotiorum, enquanto o efeito bioestimulante no crescimento do algodoeiro foi mais pronunciado com Trichoderma lentiforme CMAA 1585. Na otimização da produção de Trichoderma asperelloides na farinha de arroz, o teor de nitrogênio (0,1% p/p) e o tipo de fermentador (Erlenmeyer) tiveram efeitos significativos na obtenção de maiores rendimentos, enquanto a melhor fonte de nitrogênio foi a levedura hidrolisada (Hilyses®). As formulações GControle, GBreak-Thru, GBentonita e GComposto orgânico + Break-Thru foram as que formaram o maior número de unidades formadoras de colônia g-1 (UFC g-1) após reidratação em ágar-água. A viabilidade à temperatura ambiente foi mantida estável por até 3 meses nas formulações GControle e GBentonita, enquanto em condições refrigeradas a viabilidade foi mantida por 12 meses nas formulações GBentonita e GComposto orgânico + Break Thru. Não foram observadas diferenças significativas na inibição da germinação miceliogênica de escleródios de Sclerotinia sclerotiorum no solo pela aplicação da formulação GControle nas doses de 5 × 104, 5 × 105 ou 5 × 106 UFC g-1 de solo, mantendo um índice de controle de escleródios em 79,2; 87,5; e 93,7%, respectivamente. Desta forma, pode ser considerado que Trichoderma asperelloides CMAA 1584 apresenta a maior eficiência no controle de Sclerotinia sclerotiorum, enquanto Trichoderma lentiforme CMAA 1585 apresenta a maior promoção de crescimento das plantas, podendo a mistura de ambos ser usada para o controle do patógeno e como bioestimulante em plantas de algodão. Palavras-chave: biofungicida, biofertilizante, formulação, mofo branco e fermentação sólida ABSTRACT Fungi of the genus Trichoderma present a complex arsenal of mechanisms involved in plant protection, which include suppression of plant pathogens, growth promotion and mitigation of abiotic stresses in plants. Therefore, the selection of potential isolates must be performed carefully, as Trichoderma spp. are highly diversified in effectiveness in suppressing plant pathogens, showing varied responses according to the strains being confronted. Other challenges are related to multiplication, storage, and development of stable formulations with adequate shelf life. Overcoming these challenges will help make products of adequate quality available on the market. The objectives of this study were to select Trichoderma spp. strains growth promoters in cotton and with biocontrol activity against sclerotia of Sclerotinia sclerotiorum, to optimize the production of Trichoderma asperelloides in rice flour, and to develop granular formulations based on rice flour. Trichoderma asperelloides CMAA 1584 is more efficient in controlling Sclerotinia sclerotiorum, while the biostimulating effect on cotton growth was more pronounced with Trichoderma lentiforme CMAA 1585. In optimizing the production of Trichoderma asperelloides in rice flour, the nitrogen content (0.1% w/w) and the type of fermenter (Erlenmeyer flasks) had significant effects in obtaining higher yields, while hydrolyzed yeast (Hilyses®) was the best source of nitrogen. The formulations GControl, GBreak-Thru, GBentonite and GOrganic compost + Break-Thru were those that formed the highest number of colonies forming unit g-1 (CFU g-1) after rehydration in water-agar. Viability at room temperature was maintained stable for up to 3 months in GControl and GBentonite formulations while under refrigerated conditions, viability was maintained for 12 months in GBentonite and GOrganic compost + Break- Thru formulations. No significant differences were observed in the inhibition of the mycelogenic germination of Sclerotinia sclerotiorum in soil by the application GControl formulation at doses of 5 × 104, 5 × 105 or 5 × 106 CFU g-1 of soil, maintaining a sclerotia control index of 79.2; 87.5; and 93.7%, respectively. Thus, Trichoderma asperelloides is more efficient in controlling Sclerotinia sclerotiorum, while Trichoderma lentiforme is more suitable as a biostimulant in cotton plants. Keywords: biofungicide, biofertilizer, formulation, white mold and solid-state fermentation SUMÁRIO INTRODUÇÃO GERAL 13 CHAPTER 1 - DUAL FUNCTIONALITY OF Trichoderma: BIOCONTROL OF Sclerotinia sclerotiorum AND BIOSTIMULANT OF COTTON PLANTS 20 INTRODUCTION 22 MATERIAL AND METHODS 24 Microorganisms 25 Ability of Trichoderma strains to solubilize phosphate 26 Antifungal activity of Trichoderma strains against S. sclerotiorum 27 Parasitism of S. sclerotiorum sclerotia by Trichoderma strains 28 Germination and vigor of cotton seeds treated with Trichoderma 29 Effect of Trichoderma on cotton growth 29 Statistical analysis 30 RESULTS 30 Morphological characterization of indigenous Trichoderma spp. strains 31 Phosphate solubilization 31 Antifungal activity of Trichoderma strains against S. sclerotiorum 31 Parasitism of S. sclerotiorum sclerotia by Trichoderma strains 32 Germination and vigor of cotton seeds treated with Trichoderma 33 Effect of Trichoderma strains on cotton growth promotion 33 DISCUSSION 33 REFERENCES 39 CHAPTER 2 - BIOREACTOR-IN-A-GRANULE DESIGNED FOR Trichoderma asperelloides USING RICE FLOUR AND ITS EFFICACY AGAINST Sclerotinia sclerotiorum 52 INTRODUCTION 54 MATERIAL AND METHODS 57 Microorganisms 57 Optimization of solid-state fermentation in rice flour 58 Screening nitrogen sources 59 Mass production and formulations 59 Storage stability 60 Conidiation of T. asperelloides formulations 60 Effectiveness of T. asperelloides formulation against S. sclerotiorum 61 Statistical analysis 62 RESULTS 62 Selection of key variables for the production of T. asperelloides in rice flour 62 Screening of nitrogen sources for T. asperelloides productions in rice flour 63 Storage stability 63 Conidiation of T. asperelloides formulations 64 Effectiveness of T. asperelloides formulations against S. sclerotiorum 64 DISCUSSION 64 REFERENCES 69 CONSIDERAÇÕES FINAIS 84 REFERÊNCIAS BIBLIOGRÁFICAS 85 13 INTRODUÇÃO GERAL Aliar compromissos ambientais e a necessidade de crescente oferta de alimentos, fibras, bioenergia e uma variedade de matérias primas e produtos é apontado como um dos principais desafios do agronegócio global (GODFRAY et al., 2010). Segundo estimativas, o agronegócio contemporâneo deverá sustentar uma população mundial de cerca de oito a nove bilhões de pessoas entre 2022 e 2050 (SMITH; GREGORY, 2013; GU et al., 2021), como resultado, a demanda mundial por calorias e proteínas para este mesmo período deverá mais que dobrar (TILMAN et al., 2011). Nesse contexto, as doenças de plantas têm papel fundamental, pois são consideradas como uma das mais sérias ameaças à produção de alimentos e à segurança alimentar em todo mundo (FAO, 2017; ZAKI et al., 2020). Embora seja difícil quantificar precisamente as perdas em produtividade, segundo a Organização das Nações Unidas para a Alimentação e a Agricultura (FAO), é estimado que 20 a 40% da produção global de alimentos seja perdida anualmente devido a ação de pragas e doenças (FAO, 2017). Desse modo, visando proteger as lavouras de possíveis perdas de safra e quedas na qualidade de seus produtos, os pesticidas químicos são extensivamente utilizados nos sistemas agrícolas globais (DAMALAS, 2009). Contudo, em virtude de seus recorrentes efeitos adversos à saúde humana (VAN MAELE-FABRY et al., 2010; KIM et al., 2017), à segurança alimentar (VERGER; BOOBIS, 2013) e à manutenção da biodiversidade (BEKETOV et al., 2013) a redução de sua dependência no manejo de pragas e doenças é apontada como um dos pilares para o desenvolvimento de uma agricultura mais sustentável. Diante disso, a busca por alternativas de manejo fitossanitário menos agressivas ao meio ambiente tem crescido de interesse entre cientistas, sociedade e indústria. Dentre as medidas propostas para auxiliar nesse processo, a utilização de microrganismos tem se mostrado como uma abordagem promissora, pois é um método seguro, economicamente vantajoso, de baixo impacto no meio ambiente e na saúde humana, e com risco mínimo para organismos benéficos não alvo, como abelhas, minhocas e predadores naturais, que são alguns dos principais agentes fornecedores de serviços ecossistêmicos (GLARE et al., 2010; VAN LENTEREN et al., 2018). Além disso, muitas espécies de microrganismos possuem importantes funções ecológicas e contribuem de forma significativa no crescimento de diversas 14 culturas agrícolas (SHARMA et al., 2013; ALORI et al., 2017), participando da decomposição e mineralização dos resíduos vegetais (RICHARDSON et al., 2009; BONONI et al., 2020), aumentando a biomassa vegetal e os teores de nutrientes no solo (BONONI et al., 2020), assim como controlando diversos fitopatógenos e pragas (MANIANIA et al., 2003; ZHANG et al, 2016), os quais impactam diretamente a obtenção de maiores produtividades. De acordo com o relatório publicado pela Research and Market (2022), o mercado global de biopesticidas está projetado para crescer a uma taxa de 13,7% ao ano, saltando de um valor estimado de US$ 12,9 bilhões em 2022 para US$ 24,6 bilhões em 2027. Segundo o Business Intelligence Panel, análise realizada anualmente pela consultoria Spark Inteligência Estratégica, a comercialização de bioinsumos no Brasil cresceu 37% na safra 2020/2021 em relação à 2019/2020, e já é responsável por movimentações financeiras da ordem de R$ 1,7 bilhão. Como resultado 21% das áreas cultivadas com soja no Brasil já fazem uso de biodefensivos, totalizando aproximadamente 7,9 milhões de hectares (SPARK INTELIGÊNCIA ESTRATÉGICA, 2020). Dentre os fitopatógenos de maior importância agrícola, o fungo Sclerotinia sclerotiorum (Lib.) De Bary, agente etiológico do mofo-branco, é considerado como um dos mais devastadores e cosmopolitas, sendo capaz de infectar mais de 400 espécies de plantas (BOLAND; HALL, 1994; BOLTON et al., 2006). Apontado como a segunda doença mais importante da sojicultura no mundo (PELTIER et al., 2012), somente nos EUA, 2,8 milhões de toneladas de perdas foram estimadas entre os anos de 2010 e 2014, o que custou aos agricultores cerca de US$ 1,2 bilhão (ALLEN et al. 2017; USDA-NASS, 2017). No Brasil, maior produtor mundial do grão, a doença causa perdas significativas com epidemias de alta prevalência e severidade, especialmente em regiões com altitudes acima de 600 m (MEYER et al., 2014). Outro agravante, é que S. sclerotiorum é endêmico em aproximadamente 27% das áreas de produção de soja no Brasil (MEYER et al., 2020), o que pode resultar em perdas econômicas de até US$ 1,47 bilhão anualmente (LEHNER et al., 2017). As perdas de rendimento são causadas principalmente pela redução da quantidade e do peso dos grãos, resultante do apodrecimento dos tecidos da planta. Para cada ponto percentual de aumento da incidência de mofo-branco ocorre uma redução média na produtividade da soja de 17,2 kg ha-1, e um incremento na produção de escleródios de 100 g ha-1 (LEHNER et al., 2017). Não obstante, em razão do grande número de plantas susceptíveis ao 15 patógeno e a maioria dos cotonicultores brasileiros cultivarem o algodão na segunda safra, isto é, após a colheita da soja, a doença também tem infligido perdas na cotonicultura nacional (SILVA et al., 2019; IMEA, 2021). Dessa forma, devido a sucessão de cultivos suscetíveis ao mofo branco, a incidência da doença em áreas de ocorrência do patógeno tem aumentado consideravelmente. De acordo com a Portaria nº 5, de 21 de agosto de 2015, do Departamento de Sanidade Vegetal/Ministério da Agricultura, Pecuária e Abastecimento (DSV/MAPA), S. sclerotiorum é considerada como uma das oito pragas/doenças de maior risco fitossanitário para o Brasil, para as quais o desenvolvimento e o registro de tecnologias de controle devem ser priorizados (BRASIL, 2015). Dentre os maiores desafios no manejo da doença, a redução do número de escleródios no solo está entre os principais, haja vista sua grande persistência e alta produção de ascósporos (inóculo inicial) (ADAMS; AYERS, 1979; WILLETTS; WONG, 1980; MEYER et al., 2022). Escleródios são estruturas de resistência formados por agregados de hifas e consistem principalmente de uma camada externa com células melanizadas, o que confere alta resistência às condições ambientais adversas e a degradação química, e um componente interno formado de carboidratos (principalmente β-1,3-glucanos) e proteínas (LE TOURNEAU, 1979). Diante disso, o uso do método químico de forma não integrada a outras estratégias de manejo tem apresentado efeito limitado e inconsistente, principalmente devido às dificuldades em alcançar uma boa cobertura com fungicidas e o tempo de aplicação em relação à liberação de ascósporos (MEYER et al., 2014; MEYER et al., 2022). Além disso, a ausência de resistência genética em cultivares comerciais e a adoção de estratégias intensivas de manejo baseadas unicamente em fungicidas, resultaram no desenvolvimento de cepas resistentes para muitos ingredientes ativos (LIANG et al., 2015; MAO et al., 2018). Portanto, é imperativo explorar medidas alternativas como o controle biológico para o manejo da doença. Como mais um importante aliado no manejo do mofo branco em cultivos agrícolas, espécies do gênero Trichoderma vêm sendo utilizadas há vários anos e com sucesso. O impacto do uso do Trichoderma no manejo de S. sclerotiorum está relacionado à sua capacidade de parasitar e degradar escleródios no solo, havendo uma correlação inversamente proporcional da frequência de aplicação de Trichoderma com a viabilidade de escleródios no solo (FERRAZ; NASSER; CAFÉ- FILHO, 2011; MEYER et al., 2022). 16 Fungos do gênero Trichoderma apresentam um complexo arsenal de mecanismos envolvidos na proteção de plantas, os quais incluem micoparasitismo, competição por nutrientes, antibiose e produção de enzimas hidrolíticas (LORITO et al., 2010; DRUZHININA et al., 2011; HERMOSA et al., 2012; MONTE et al., 2019). Além disso, devido à plasticidade de seus genomas em expressar múltiplas funções ecológicas, várias espécies de Trichoderma promovem o crescimento de plantas (RUBIO et al. 2017; MONTE et al. 2019), contribuem para a melhor utilização de nutrientes (HARMAN 2011; DOMÍNGUEZ et al. 2016) e induzem respostas de defesa contra estresses bióticos e abióticos (HERMOSA et al. 2012; BROTMAN et al. 2012; RUBIO et al. 2017; MONTE et al. 2019). As espécies/isolados de Trichoderma são altamente diversificados em eficácia na supressão de patógenos de plantas (HARMAN et al., 2004, VERMA et al., 2007), apresentando respostas variadas de acordo com as cepas que estão sendo confrontadas (ATANASOVA et al., 2013). Desse modo, a seleção dos isolados consiste no primeiro passo no desenvolvimento de produtos à base deste antagonista. Além disso, atributos como virulência, persistência e tolerância à estresses abióticos (temperatura, umidade e radiação UV), assim como baixas exigências nutricionais, alta produção de propágulos infectivos e capacidade de se desenvolver em substratos simples e baratos, são de extrema importância, pois o conceito de produção em massa se baseia nas necessidades de uso inundativo, logo requerem um elevado número de propágulos a fim de atingir o alvo ou colonizar o habitat (FARIA; WRAIGHT, 2001). A produção massal de Trichoderma spp. pode ser realizada de três formas: via fermentação sólida, líquida ou bifásica (MASCARIN et al., 2019). A primeira delas, também conhecida como fermentação semi-sólida ou fermentação sólida estática, o crescimento microbiano ocorre na ausência de água livre, ou seja, a umidade necessária ao seu crescimento se encontra absorvida ou complexada no interior da matriz sólida (LONSANE et al. 1985; SOCCOL 1996). Enquanto na fermentação líquida ou submersa, como o próprio nome sugere, o crescimento microbiano é realizado em soluções nutritivas líquidas (JACKSON, 1997). No Brasil, a maioria das biofábricas utiliza o sistema de fermentação bifásica, na qual o inóculo é inicialmente produzido em cultura líquida e, posteriormente, transferido para substratos sólidos para a produção de conídios aéreos (KUMAR et al., 2007; LI et al., 2010; MASCARIN et al., 2010; WOO et al., 2014; MASCARIN et al., 2019). Nesse processo, grãos de arroz são majoritariamente utilizados como substrato 17 e se realiza a incubação em sacos de polipropileno ou em bandejas por um período de 10 a 14 dias, com posterior remoção dos conídios (FARIA; WRAIGHT, 2007; LI et al., 2010; MASCARIN et al., 2019). No entanto, devido às características hidrofílicas dos conídios aéreos de Trichoderma (JIN; CUSTIS, 2011) e a necessidade de extrair os conídios dos grãos, muitos fabricantes lavam os substratos colonizados com soluções surfactantes antes da formulação para concentrar a biomassa (FARIA; WRAIGHT, 2007; LI et al., 2010; MASCARIN et al., 2019). Entretanto, ao longo deste processo, os metabólitos que possuem propriedades antimicrobianas e/ou atuam como estimulantes vegetais são inevitavelmente perdidos. Adicionalmente, o resíduo sólido após a extração dos esporos necessita de destinação adequada, sendo geralmente explorada na produção de energia (Elias et al., 2022) ou compostagem. Para contornar parte desse problema e reduzir os custos de produção, tendo em vista que os substratos podem representar mais de 50% dos custos de produção (ELTEM et al., 2014; STANBURY et al., 2017), diversos subprodutos e resíduos agroindustriais são frequentemente avaliados em processos fermentativos, pois são abundantes e muitas vezes subutilizados (FARINAS, 2015; SOCCOL et al., 2017). Além disso, a reutilização de subprodutos agroindustriais para a geração de novos produtos de alto valor agregado é extremamente benéfica e fomentada internacionalmente, como pode ser observado no plano de ação da União Europeia para uma economia circular (COMISSÃO EUROPEIA, 2020). O arroz (Oryza sativa L.) é uma das principais culturas de cereais, bem como alimento básico para quase metade da população mundial, especialmente nos países asiáticos (BIRD et al., 2000). Contudo, até chegar à mesa do consumidor, uma série de processos são empregados para o beneficiamento do grão, os quais combinados produzem diversos subprodutos (ESA et al., 2013). O arroz quebrado, um dos subprodutos do beneficiamento do grão, representa cerca de 10-15% do arroz beneficiado (NUNES et al., 2017) e é comercializado por 30-50% do valor do grão inteiro (NUNES et al., 2017; LI et al., 2019), sendo pouco aproveitado para a alimentação humana e majoritariamente utilizado para a alimentação animal (NUNES et al., 2017). Assim, devido ao seu baixo custo, alta disponibilidade e valor nutricional (74% amido e 7% proteína) (LIU et al., 2016), várias tecnologias têm sido propostas para aumentar seu uso na indústria (AHMED et al., 2015; BICH et al., 2018; MYBURGH et al., 2019; NAKANO et al., 2012). Dentre elas, o uso como substrato para produção 18 massal e como inerte em formulações de Trichoderma tem potencial em garantir bons sistemas de entrega e proporcionar vantagens competitivas em relação à comunidade nativa do solo, uma vez que o Trichoderma spp. pode hidrolisar o amido em açúcares simples e de metabolização rápida (GUIMARÃES et al., 2018; KLAIC et al., 2018) para sua nutrição. Além disso, melhorias na viabilidade e vida de prateleira de microrganismos formulados com compostos amiláceos são relatadas, em razão de seu suporte estrutural e proteção contra estresses térmicos, oxidativos e osmóticos (CHAN et al., 2011; SCHOEBITZ et al., 2012; TAL et al., 1999). Assim, a moagem do arroz quebrado em farinha pode se tornar uma alternativa simples, barata e livre de resíduos para o desenvolvimento de novos produtos à base de Trichoderma, além de remover a etapa de extração de conídios e manter os metabólitos no produto final. Contudo, para maximizar os rendimentos, a otimização das condições de cultivo é imprescindível, pois a descoberta das condições que levam a uma esporulação mais rápida, pode ser o fator mais importante na redução dos custos de produção (JACKSON, 1997). Além dos métodos de produção, as formulações desempenham um papel fundamental na determinação do sucesso de um produto. Os componentes de uma formulação geralmente são categorizados em três partes: ingrediente ativo, veículo e adjuvantes (ASH, 2010; BURGES, 1998). O ingrediente ativo é a forma infecciosa do microrganismo (conídios, microescleródios, blastósporos, endósporos, micélios, etc.); os veículos são inertes utilizados para diluir o agente ativo; e os adjuvantes compreendem uma ampla variedade de agentes que aprimoram uma ou mais características da formulação (BURGES, 1998). Segundo Lewis e Papavizas (1985), a adição de amido como veículo em formulações de Trichoderma viride, Trichoderma harzianum e Trichoderma hamatum aumentou em até 100 vezes o número de UFC g-1 de solo. Portanto, há evidências que sustentam a hipótese de que a utilização de farinha de arroz para a produção de Trichoderma, seguida pela formulação via extrusão/granulação, tem potencial em fornecer condições adequadas ao estabelecimento do Trichoderma no solo, uma vez que, o grânulo atuará como um mini reator ao seu crescimento inicial. A apresentação dos estudos desenvolvidos nesta tese de doutorado está estruturada em dois capítulos, organizados da seguinte forma: no capítulo 1 são apresentados os resultados obtidos na seleção dos isolados, na qual foram avaliados a eficiência no parasitismo a Sclerotinia sclerotiorum, na germinação e o vigor de 19 sementes de algodão, na promoção de crescimento do algodoeiro e na solubilização de fosfato. Esse primeiro capítulo está publicado na Frontiers in Plant Science: Silva L.G., Camargo R.C., Mascarin G.M., Nunes P.S.O., Dunlap C., Bettiol W. Dual functionality of Trichoderma: Biocontrol of Sclerotinia sclerotiorum and biostimulant of cotton plants. Frontiers in Plant Science, 13:983127, 2022. doi: 10.3389/fpls.2022.983127 No capítulo 2 são apresentados os resultados da otimização da produção de Trichoderma asperelloides em farinha de arroz, no desenvolvimento de formulações granulares e na avaliação da vida de prateleira em condições refrigeradas e à temperatura ambiente, eficiência de biocontrole a escleródios de Sclerotinia sclerotiorum, e conidiação. Este capítulo é intitulado: Bioreactor-in-a-granule designed for Trichoderma asperelloides using rice flour and its efficacy against Sclerotinia sclerotiorum 20 CHAPTER 1 DUAL FUNCTIONALITY OF Trichoderma: BIOCONTROL OF Sclerotinia sclerotiorum AND BIOSTIMULANT OF COTTON PLANTS Lucas Guedes Silva1,2, Renato Cintra Camargo2, Gabriel Moura Mascarin2*, Peterson Sylvio de Oliveira Nunes2,3, Christopher Dunlap4, Wagner Bettiol2 1 Department of Plant Protection, School of Agriculture, São Paulo State University, 18610- 034, Botucatu, SP, Brazil 2 Embrapa Environment, SP 340 Road, Km 127.5, 13918-110, Jaguariúna, SP, Brazil 3 Department of Phytopathology, Federal University of Lavras, 37200-900, Lavras, MG, Brazil 4 United States Department of Agriculture, National Center for Agricultural Utilization Research, Crop Bioprotection Research Unit, 1815 North University St, Peoria, IL 61604, USA *Corresponding author: Gabriel Moura Mascarin – gabriel.mascarin@embrapa.br Reference: Silva L.G., Camargo R.C., Mascarin G.M., Nunes P.S.O., Dunlap C., Bettiol W. Dual functionality of Trichoderma: Biocontrol of Sclerotinia sclerotiorum and biostimulant of cotton plants. Frontiers in Plant Science, 13:983127, 2022. doi: 10.3389/fpls.2022.983127. Graphical Abstract mailto:gabriel.mascarin@embrapa.br 21 Abstract Microbial crop protection products based on Trichoderma have the ability to display multifunctional roles in plant protection, such as; pathogen parasitism, enhance nutrient availability and stimulate plant growth, and these traits can be used to enhance the overall agronomic performance of a variety of crops. In the current study, we explored the multifunctional potential of two indigenous Brazilian strains of Trichoderma (T. asperelloides CMAA 1584 and T. lentiforme CMAA 1585) for their capability of controlling Sclerotinia sclerotiorum, a key plant pathogen of cotton, and for their ability of growth promotion in cotton plants (Gossypium hirsutum). Both strains were able to solubilize mineral phosphorus (CaHPO4), to release volatile organic compounds that impaired the mycelial growth of S. sclerotiorum, and to promote the growth of cotton plants under greenhouse conditions. In dual culture, Trichoderma strains reduced the growth rate and the number of sclerotia formed by S. sclerotiorum. By treating sclerotia with conidial suspensions of these Trichoderma strains, a strong inhibition of the myceliogenic germination was observed, as a result of the marked mycoparasitic activity exerted on the sclerotia. The parasitism over S. sclerotiorum was more effective with T. asperelloides CMAA 1584, whereas the effect of biostimulants on cotton growth was more pronounced with T. lentiforme CMAA 1585 that also showed a higher capacity of phosphate solubilization. Thus, T. asperelloides CMAA 1584 displays higher efficiency in controlling S. sclerotiorum, while T. lentiforme CMAA 1585 is more suitable as a biostimulant due to its ability to promote cotton plants growth. Overall, these Trichoderma strains may be used in mixture to provide both pathogen control and promotion of plant growth, and this strategy will support growers in minimizing the use of synthetic fertilizers and fungicides against white mold in cotton crops. Keywords: Bioprotectant; biofungicide; white mold; biofertilizer; phosphate solubilization ability. 22 INTRODUCTION Cotton (Gossypium hirsutum L.) is the most important source of natural fibers in the world (Sivakumar et al., 2021; Wu et al., 2022). According to Tarazi et al. (2019), approximately 150 countries are directly involved in the cotton industrial chain, being an income source for more than 100 million families worldwide. Brazil stands out as the fourth largest producer of cotton worldwide, attaining a cultivated area of 1.6 million hectares with an estimated crop production of 6.7 million tons in 2021/22 (CONAB, 2022). Since 1990s, the cotton growing area has dramatically expanded throughout the savannah Central-West region of Brazil (known as biome ‘Cerrado’), mainly due to breeding efforts for developing locally adapted high-yielding cultivars and improving agronomic practices (Morello et al., 2015; Silva Neto et al., 2016; Barroso et al., 2017; Silva et al., 2019). As a result, the cultivated area in the Mato Grosso State has increased by approximately 1,500% in the last 30 years (ABRAPA, 2021). However, the high incidence of pests and diseases remain inflicting high productivity losses, accounting for approximately 35% of production costs (IMEA, 2019). Among several diseases that limit cotton growth and yield, white mold, also known as Sclerotinia stem rot, caused by the ascomycete fungus Sclerotinia sclerotiorum (Lib.) de Bary (Ascomycota: Sclerotiniaceae), is one of the most devastating and yield-limiting diseases. This plant pathogen cause billions of dollars of crop losses and is of great economic importance to several agricultural and vegetable crops worldwide, notably including cotton and soybean (Boland and Hall, 1994; O’Sullivan et al., 2021). According to the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), S. sclerotiorum is considered one of the eight diseases/pests with the highest phytosanitary risk for Brazil (Brazil, 2015). This disease poses a serious threat to cotton plants at all phenological stages, and pathogen forms a dark-pigmented and hardened mycelial threads known as sclerotium capable of surviving for several years in 23 soil (Tourneau, 1979; Schwartz and Singh, 2013). Symptoms associated with the white mold in cotton include wilt, necrosis and rotting of stems, bolls, petioles and leaves (Charchar et al., 1999; Suassuna et al., 2019). Currently, the Mato Grosso State is responsible for approximately 71% of the Brazilian cotton production, following an integrated cropping system with soybean and corn (Silva et al., 2019; IMEA, 2021). According to Meyer et al. (2020), S. sclerotiorum is endemic in approximately 27% of soybean production areas in Brazil, and 87% of cotton growing areas in Mato Grosso, in which cotton is cultivated as a second crop after soybean crop (IMEA, 2021). Thus, the succession of susceptible crops to S. sclerotiorum has been responsible for the continuous increase of the incidence of white mold, leading to an overuse of chemical fungicides as a means to alleviate crop yield losses. However, over-reliance of broad-spectrum chemical fungicides poses a serious risk to the environment (Komárek et al., 2010), health of growers (Kniss, 2017), and accelerate the selection of resistant strains of S. sclerotiorum (Zhou et al., 2014), all of which requires urgent alternative measures that include the development bio-rational solutions for the integrated management of white mold disease. Despite the fact that chemical fungicides are effective in protecting plants from the white mold, their stand-alone use has inconsistent and unsatisfactory results. This is mainly due to difficulties in achieving adequate application coverage of the target pathogen, coupled with the best timing of application when ascospores are discharged (Meyer et al., 2014). Additionally, the lack of genetically resistant plants and the adoption of intensive management strategies based solely on synthetic fungicides have resulted in the development of resistant S. sclerotiorum strains to many chemical active ingredients (Liang et al., 2015; Mao et al., 2018). In this sense, it is imperative to explore alternative measures such as biological control strategies against this cosmopolitan plant pathogen (Bettiol et al., 2021). 24 Among biological control agents, Trichoderma spp. are considered effective in controlling S. sclerotiorum across several crops (Li et al., 2005; Sharma and Sain, 2010; Elias et al., 2016; Sumida et al., 2018), including cotton. The efficacy of using the necrotrophic mycoparasite Trichoderma in the management of S. sclerotiorum is related to its ability to parasitize and degrade sclerotia, resulting in an inversely proportional relationship between the frequency of Trichoderma application and the viability of sclerotia in the soil (Ferraz et al., 2011; Geraldine et al., 2013; Smolińska and Kowalska, 2018). Notably, Trichoderma spp. antagonize a myriad of plant pathogens by distinct mechanisms of action (Lorito et al., 2010; Druzhinina et al., 2011; Hermosa et al., 2012; Monte et al., 2019; Monte and Hermosa, 2021). Furthermore, due to the plasticity of their genomes in expressing multiple ecological functions and diverse biochemical machinery, several Trichoderma species promote plant growth (Rubio et al., 2017; Monte et al., 2019; Monte and Hermosa, 2021) and induce plant defenses against biotic and abiotic stresses (Brotman et al., 2012; Hermosa et al., 2012; Rubio et al., 2017; Monte et al., 2019). The effectiveness of plant pathogen suppression as well as growth promotion mediated by Trichoderma are species and strain dependent (Harman et al., 2004; Verma et al., 2007; Atanasova et al., 2013; Haddad et al., 2017; Sumida et al., 2018). Owing to the lack of studies exploring the biocontrol and biostimulant abilities of Trichoderma strains in association with cotton plants, this study aimed to investigate the potential of two novel indigenous Brazilian strains, Trichoderma asperelloides CMAA 1584 and Trichoderma lentiforme CMAA 1585, against S. sclerotiorum along with their role as cotton growth promoters. MATERIAL AND METHODS 25 Microorganisms Trichoderma asperelloides CMAA 1584 (BRM 065723, GenBank accession ON542481) and Trichoderma lentiforme CMAA 1585 (BRM 065775, GenBank accession ON542480), both isolated from soil in Jaguariúna, SP, Brazil (22º43'43" S and 47º01'04" W), and deposited in the Collection of Microorganisms of Agricultural and Environmental Importance (CMAA) from Embrapa Environment (Jaguariúna, SP, Brazil), were used in these studies. These strains were reactivated and grown on potato-dextrose-agar medium (PDA; Acumedia Manufacturers®, Michigan, USA) in Petri dishes (9 × 1.5 cm) for 14 days at 25 ± 2 ºC and 12:12 hours photoperiod. For preservation, 7-day-old sporulated colonies grown on PDA were cut into 5 mm pieces, placed in cryovials containing 1.5 mL of sterile solution of 20% (v/v) glycerol (Dinâmica®, São Paulo, SP, Brazil) prepared with double deionized water, and stored at –80 ºC as stock cultures. Five-day-old PDA-grown cultures of these two Trichoderma strains were morphologically characterized based on colony growth aspects, conidiophores, and conidia size. Conidia size measurements were recorded with a light phase-contrast microscope (Olympus CS43 microscope and Olympus EP50 camera). The strains were identified phylogenetically using the translation elongation factor 1-α gene through direct comparison with data from reference type strains. The plant pathogen Sclerotinia sclerotiorum CMAA 1105 (GenBank accession OM348513) was cultured on PDA in Petri dishes through myceliogenic germination from surface-sterilized sclerotia, and the newly-formed sclerotia were stored at 4 ºC. This S. sclerotiorum strain was isolated in Jaguariúna, SP, Brazil (22º43'43" S and 47º01'04" W) in 1992, and was then deposited in the Collection of Microorganisms of Agricultural and Environmental Importance (CMAA) from Embrapa Environment (Jaguariúna, SP, Brazil). All fungal strains used in this study are registered under the Brazilian genetic heritage – SisGen – protocol A135E26. 26 Ability of Trichoderma strains to solubilize phosphate The ability of Trichoderma strains to solubilize inorganic phosphate (P) was evaluated by quantifying the solubilized P in liquid NBRIP (National Botanical Research Institute's Phosphate) medium, which contained per liter: 10.0 g glucose, 5.0 g MgCl2.6H2O, 0.25 g MgSO4.7H2O, 0.2 g KCl and 0.1 g (NH4)2SO4 (Nautiyal, 1999). In the medium, 50 mL of K2HPO4 (10%) and 100 mL of CaCl2 (10%) were added to form an insoluble calcium phosphate (CaHPO4) precipitate. For inoculum production, 7-day-old sporulated cultures of each Trichoderma strain were rinsed with 10 mL of a sterile solution containing 0.04% polyoxyethylene sorbitan mono-oleate (Tween® 80, Synth, SP, Brazil) and calibrated using a hemocytometer (improved Neubauer chamber, 400× magnification) under a microscope (DM 500, Leica Microsystems GmbH®, Germany) to provide a final inoculum size of 5 × 106 conidia mL-1 in the medium. These liquid cultures were then incubated at 28 ± 1 °C in an orbital rotary shaker (TE-1401, Tecnal®, Piracicaba, SP, Brazil) at 180 rpm for 5 days with a 12:12 hours photoperiod. The amount of calcium phosphate in the medium before inoculation of Trichoderma strains were approximately 150 µg mL-1. Aliquots of 1 mL were taken at the 5th day and centrifuged at 7,000 rpm and 22 ºC for 5 minutes to determine the concentration of soluble phosphorus, according to the colorimetric method described by Murphy and Riley (1962). The concentration of solubilized P in the supernatant was calibrated based on a standard curve of CaHPO4 (Sigma-Aldrich®, St. Louis, MO, USA) at concentrations of 0.5, 1.0, 2.0, 2.5, and 5.0 mg mL-1. The experiments were carried out with four biological repetitions to each fungal strain. Untreated control group (blank) was performed without the presence of microorganisms, whose values obtained were subtracted from those obtained in the presence of the fungal inoculum as a means to normalize the absorbance reads. 27 Antifungal activity of Trichoderma strains against S. sclerotiorum The ability of Trichoderma strains to antagonize S. sclerotiorum was evaluated by dual culture tests. Mycelial plugs (5 mm diameter) from the colony margin of an actively growing Trichoderma culture in PDA were placed on the edge of the Petri dish, and another plug of 7- day-old colony of S. sclerotiorum cultured on PDA was placed on the opposite side, maintaining 7 cm apart from colony discs. The plates were incubated at 25 ± 2 ºC and the mycelial growth of both fungi was measured daily until Trichoderma strains have overgrown or surrounded the S. sclerotiorum colony. After 14 days of incubation under dual culturing, the antagonistic potential of Trichoderma strains inhibiting the pathogen's growth was measured and the development of sclerotia was also evaluated. Furthermore, a diagrammatic scale proposed by Bell et al. (1982) was used to score the antagonistic capacity, where: 1 - Trichoderma overcomes the pathogen and grows in 100% of the plate; 2 - Trichoderma grows on at least 75% of the plate; 3 - Trichoderma and the pathogen colonize approximately 50% of the plate; 4 - The pathogen colonizes at least 75% of the plate and resists to Trichoderma; 5 - The pathogen completely overlaps Trichoderma and occupies the entire surface of the plate. As a control, Petri dishes inoculated only with the pathogen served as the reference to calculate the percent inhibition of pathogen’s colony growth exerted by Trichoderma strains. The experiment was performed with five biological replicates for each strain. To assess the effect of volatile organic compounds (VOCs) released by Trichoderma strains on S. sclerotiorum mycelial growth, two Petri dish bottoms, one containing the pathogen and the other with a Trichoderma strain, all plated in the center and grown on PDA, were superimposed (Muthukumar et al., 2011). As a control, Petri dishes containing the pathogen were overlaid with another containing only PDA. These paired cultures were maintained in a growth chamber under the same environmental conditions described above. After 2 days of incubation, due to the rapid mycelial growth of S. sclerotiorum, the percentage of inhibition of 28 the pathogen was assessed and further calculated by the equation: Inhibition (%) = (D1 – D2)/D1 × 100, where D1 represents the radial diameter of the pathogen in the control treatment, and D2 the radial diameter of the pathogen confronted with Trichoderma. The experiment was performed with five biological replicates for each strain. Parasitism of S. sclerotiorum sclerotia by Trichoderma strains The ability of both Trichoderma strains in parasitizing S. sclerotiorum sclerotia was evaluated in polypropylene boxes (11 cm × 11 cm × 3.5 cm) (Gerbox®) containing 200 g of a dystroferric dark red latosol, collected at Embrapa Environment and autoclaved at 121 ºC for 60 minutes on three consecutive days. Dark-pigmented mature S. sclerotiorum sclerotia were produced in 500 mL Erlenmeyer flasks containing carrot and cornmeal, according to Garcia et al. (2012). Each autoclaved flask received three 5-mm-PDA discs of S. sclerotiorum mycelium, taken from the edge of a 7-day-old colony and incubated at 25 ± 2 ºC. After 30 days of growth on carrot-cornmeal substrate, mature sclerotia were removed, placed on absorbent paper inside a laminar flow chamber, left drying for 24 hours, and then kept in a refrigerator at 4 °C prior to using in bioassays. In each polypropylene box, 12 sclerotia were randomly distributed on the soil surface, and 10 mL suspensions containing 1 × 106, 1 × 107, and 1 × 108 conidia mL-1 of each Trichoderma strain were evenly applied with a pipette over the soil surface. All groups were incubated for 15 days at 25 ± 2 ºC with a photoperiod of 12:12 hours (Geraldine et al., 2013). A control group was set up with sterile distilled water. After 15 days of incubation, all sclerotia were removed from the soil, surface-sterilized with ethanol (70%) and sodium hypochlorite (2%) for 2 minutes, and subsequently rinsed three times in sterile distilled water prior to plating them on a selective media. Soft and disintegrated sclerotia due to colonization by Trichoderma strains were counted after slight pressure with a tweezer (Henis et al., 1983). Sclerotia viability was evaluated by incubating them on Neon medium (Napoleão et al., 2006) 29 for 7 days at 25 ± 2 ºC, then observing for the formation of a yellow halo around the sclerotia, which were deemed to be viable. The experiment was performed in a completely randomized design for each strain, with three treatments (inoculum size) and four biological replicates, in addition to a mock control treated only with water. Germination and vigor of cotton seeds treated with Trichoderma Seeds of cotton cv. FM 975 WS® provided by Instituto Mato-Grossense do Algodão (IMA, Mato Grosso, Brazil) were used in the interaction studies involving Trichoderma strains and cotton plants. The seeds were surface disinfected in 70% ethanol followed by 2% sodium hypochlorite solution for 2 minutes and washed in sterile distilled water three times. Surface- sterilized cotton seeds were soaked in an aqueous Trichoderma suspension containing 1 × 106, 1 × 107, and 1 × 108 conidia mL-1 for 60 minutes, and then layered on a Petri dish to air-dry for 1 hour inside a laminar flow hood. Trichoderma-treated cotton seeds were sown in germitest® paper (Cienlab Equipamentos Científicos Ltda, Campinas, SP, Brazil) using a roller system moistened with distilled water and incubated at 25 ± 2 ºC. The experiment was set up in a completely randomized design with three treatments (inoculum concentrations) and four independent biological replicates, with 20 seeds each (i.e., total of 80 seeds per treatment). The number of germinated seeds was determined on the 4th day after sowing, being expressed as a percentage of germinated seeds. Afterwards, the seedlings were dried in an oven at 105 ºC until constant weight, and the vigor index was determined according to Abdul‐Baki and Anderson (1973) by the equation: Vigor Index = Germination (%) × Seedling dry weight (g). Effect of Trichoderma on cotton growth Cotton seeds cv. FM 975 WS® were treated with crescent concentrations of Trichoderma conidia as described above and sown in rhizotron made of polyvinyl chloride 30 (PVC) half-longitudinal tubes (100 cm height × 17.5 cm diameter), containing a mixture of a dystroferric dark red latosol and sand in a ratio of 2:1 (v/v). The soil exhibited the following chemical and physical attributes analyzed at 0 - 20 cm depth: pH in H2O = 4.3; OM = 32.3 g kg-1; P = 9.36 mg dm-3; Ca = 3.09 cmolc dm-3; Mg = 1.48 cmolc dm-3; K = 128.55 mg dm-3; SB = 4.95 cmolc dm-3; H + Al = 6.10 cmolc dm-3; t = 4.99 cmolc dm-3; V% = 44.54. In addition to seed treatment, 10 mL of the same conidial suspensions were applied in the planting furrow via drench at 15, 30 and 45 days after sowing (DAS). The experiment was set up in a randomized block design with three treatments (inoculum concentrations) and five independent biological replicates, in addition to mock cotton seeds as a control. The assay was carried out in a greenhouse for 60 days and the following growth parameters of the cotton plants were evaluated: height (14, 24, 31 and 55 DAS), root length (at 7, 14, 24 and 60 DAS) and leaf area of the first non-cotyledonary leaf (at 24 DAS), as described by Grimes and Carter (1969). At 60 DAS the following parameters were determined: stem diameter (2 cm above the soil surface), fresh and dry weights for both the aboveground portion and roots of the plants. Statistical analysis Homogeneity of variances and normality tests were performed by Bartlett’s and Shapiro-Wilk tests. Data were fitted to linear models and analyzed by analysis of variance (ANOVA) using original data sets to identify significant differences between means of the treatments (Tukey’s test, P < 0.05). Statistical analyses were performed using Minitab® software version 19.1. RESULTS 31 Morphological characterization of indigenous Trichoderma spp. strains According to the phylogenetic analysis based on tef-alpha 1 gene, the strain CMAA 1584 was confirmed to be Trichoderma asperelloides, while the strain CMAA 1585 was identified as Trichoderma lentiforme. Purified monosporic cultures of these two Trichoderma spp. strains were very divergent from each other in terms of growth, color, conidial size, and conidiophores. As depicted in Figure 1, cultures of T. asperelloides CMAA 1584 exhibited profuse growth on PDA with dark green color when fully sporulated and forming ovoid conidia averaging 3.60 × 3.59 µm (length and width) with a resultant area estimated in 10.10 µm2 (standard error: ± 0.17 µm2, n = 20). When looking at T. lentiforme CMAA 1585 cultures, its sporulated colony assumed pale greenish color and produced conidia averaging 2.54 × 2.44 µm (length and width) with an estimated average area of 4.88 µm2 (standard error: ± 0.18 µm2, n = 20). The area size of T. asperelloides was noted to be twice larger than conidia of T. lentiforme. The morphological phenotypes and conidia sizes are consistent with values previously reported for these species (Samuels et al., 2010; Chaverri et al., 2015). Phosphate solubilization Trichoderma lentiforme CMAA 1585 and T. asperelloides CMAA 1584 were both capable of solubilizing inorganic phosphate, resulting in about 31.7% and 5.2% of CaHPO4 remaining in the medium, respectively, in comparison to control (Table 1). Phosphate solubilization was significantly (P < 0.05) higher in NBRIP medium, in which T. lentiforme solubilized significantly more phosphate than T. asperelloides (Table 1). Antifungal activity of Trichoderma strains against S. sclerotiorum In general, volatile organic compounds (VOCs) released by Trichoderma strains significantly reduced (P < 0.05) the mycelial growth of S. sclerotiorum (Table 2). Compared to 32 the control, VOCs emitted by T. lentiforme CMAA 1585 and T. asperelloides CMAA 1584 significantly reduced the growth rate of S. sclerotiorum by 55% and 53%, respectively (Table 2). However, there was no difference between these Trichoderma strains in their ability to inhibit this pathogen by means of released VOCs. In dual culture assay for direct confrontation, T. asperelloides CMAA 1584 and T. lentiforme CMAA 1585 reduced the growth rate (mm day-1) of S. sclerotiorum in 10% and 13%, respectively, when compared to control (Table 2) (P < 0.05). The inhibition of mycelial growth was 9.5% and 12.2% for T. asperelloides CMAA 1584 and T. lentiforme CMAA 1585, respectively (Table 2). Notably, T. asperelloides CMAA 1584 and T. lentiforme CMAA 1585 remarkably decreased by 96% and 47% the number of sclerotia formed by S. sclerotiorum colony in comparison to control, respectively (Table 2, P < 0.05). Trichoderma lentiforme CMAA 1585 received higher scores (3.6) when compared to T. asperelloides CMAA 1584 (2.2) according to Bell’s diagrammatic scale, indicating that the former was less aggressive in parasitizing sclerotia than the latter (Figure 2). Parasitism of S. sclerotiorum sclerotia by Trichoderma strains The myceliogenic germination of sclerotia was significantly (P < 0.05) reduced by both Trichoderma strains (Figure 3). All concentrations of T. asperelloides CMAA 1584 colonized 100% of sclerotia and thus strongly inhibited the myceliogenic germination of all sclerotia (Figure 3A). Trichoderma lentiforme CMAA 1585, despite colonizing 100% of sclerotia, only 69% sclerotia were found ungerminated or non-viable based on the Neon selective medium test. The degradation of sclerotia did not reveal significant differences (P > 0.05) between Trichoderma strains for all concentrations tested (Figure 3B). 33 Germination and vigor of cotton seeds treated with Trichoderma Trichoderma asperelloides CMAA 1584 and T. lentiforme CMAA 1585 applied in cotton seeds, at 1 × 106, 1 × 107, and 1 × 108 conidia mL-1, did not show any significant differences (P > 0.05) for seed germination and initial vigor of cotton seedlings, when compared to control (Table 3). Effect of Trichoderma strains on cotton growth promotion Under greenhouse conditions, growth of cotton plants derived from seeds coated with spores of T. asperelloides CMAA 1584 and T. lentiforme CMAA 1585 strains were compared with mock control plants. Notably, T. lentiforme CMAA 1585 outperformed T. asperelloides CMAA 1584 in promoting growth of cotton plants (Tables 4 and 5, Figures 4 and 5). Looking at T. asperelloides CMAA 1584, this strain incited cotton growth promotion only for leaf area (P < 0.05), reaching indexes of 98.9% and 42.0% higher than the mock control plants, when applied to seeds at 1 × 107 and 1 × 108 conidia mL-1, respectively (Table 4, Figure 5AB). Plant height, stem diameter, aboveground and root fresh and dry weights increased (P < 0.05) with the application of T. lentiforme CMAA 1585 at 1 × 108 conidia mL-1 (Table 5, Figure 4). Notably, cotton plants derived from seeds treated with T. lentiforme CMAA 1585 at 1 × 108 conidia mL-1 increased stem diameter, height, aboveground and root fresh and dry weights of 23.7%, 35.2%, 69.3%, 86.7%, 46.0%, and 30.4% (Table 5, Figures 4 and 5), when compared to control plants (P < 0.05), respectively. DISCUSSION The present study reveals the ability of two indigenous Brazilian strains, T. lentiforme CMAA 1585 and T. asperelloides CMAA 1584 to solubilize inorganic phosphorus, a macronutrient of low availability in tropical soils. Furthermore, these strains are capable of 34 emanating VOCs which inhibit the mycelial growth of S. sclerotiorum. In addition, these strains reduce the growth rate and total number of sclerotia of S. sclerotiorum in dual culture assay, and they inhibit the myceliogenic germination due to degradation of sclerotia through direct parasitism. The difference in biocontrol performance between the T. lentiforme CMAA 1585 and T. asperelloides CMAA 1584 lies in their ability to suppress the myceliogenic germination of sclerotia, as reported in the present study (Figure 2), which may be related to secondary metabolites, including antifungal compounds, and the direct capacity of parasitism using an arsenal of well-known cuticle-degrading enzymes, where both mechanisms have been correlated with the virulence strategies employed by Trichoderma species (Harman et al., 2004; Geraldine et al., 2013; Monte et al., 2019). Previous studies have shown that T. harzianum, T. koningii, T. pseudokoningii, T. koningiopsis, T. asperellum, T. atroviride, and T. virens displayed excellent inhibitory effect on the myceliogenic germination of S. sclerotiorum in the range of 62% to 100%, when applied directly to the sclerotia (Haddad et al., 2017; Sumida et al., 2018). As noted in our study, evidence of interspecific variation in biocontrol efficacy among Trichoderma spp. is common and should be a key criterion to be incorporated into screening studies for biocontrol of plant pathogens. There is a tremendous diversity among Trichoderma species and strains in their ability to produce and release biogenic volatile organic compounds (BVOCs) with remarkable roles in mediating plant growth and antagonism towards plant pathogens (Siddiquee et al., 2012; Contreras-Cornejo et al., 2014; Li et al., 2018). In this study, we noted that both of our Trichoderma strains imposed similar detrimental effects on S. sclerotiorum growth under in vitro conditions through emission of VOCs, whose compounds remain elusive. Given the importance of some VOCs emitted by Trichoderma playing pivotal roles in plant growth and biocontrol activity against plant pathogens, further research is needed to elucidate the emission 35 profiles of VOCs by these T. asperelloides and T. lentiforme strains in view of providing new insights and applications of their metabolites in cotton growth enhancement and protection against white mold disease. Trichoderma asperelloides CMAA 1584 showed a great potential for use in biological control of S. sclerotiorum as it inhibited 100% myceliogenic germination of all sclerotia exposed to their conidia, as well as decreasing the sclerotia formation by 96.1%, when compared to the mock control. According to Atanasova et al. (2013), Trichoderma spp. craft distinct strategies to combat and outcompete other host fungi. These authors observed host sensing in T. atroviride and T. virens through expression of genes involved in the attack, whereas T. reesei was keener to outcompete the pathogen for nutrients. Thus, screening studies for potential biocontrol candidates of Trichoderma can reveal interesting phenotypical traits between species and strains and differential pattern of gene expression linked to biocontrol during the parasitism process of targeted hosts (Atanasova et al., 2013; Troian et al., 2014). Our results strengthen the need to select the antagonist strain according to the desired targeted pathogen taking into account its biology and epidemiology in the crop system (Köhl et al., 2011; Bettiol et al., 2021). Sclerotia that failed myceliogenic germination and were colonized by Trichoderma were classified as unviable, as this was the same criterion employed by Abdullah et al. (2008) and Görgen et al. (2009). Such mycotrophic lifestyle is one of the most remarkable antagonistic mechanisms expressed by Trichoderma spp. and is implicated in the direct attack of one fungal species to another (Sood et al., 2020). In this sequential process, the first step involves recognition by chemical cues of the targeted pathogenic fungus by Trichoderma, which then its hyphae attach and coil around the prey fungal hyphae (Harman et al., 2004), followed by the onset production of lytic enzymes that cause the dissolution of fungal cell walls (Druzhinina et al., 2011). Hence, it may be expected that antagonists with increased secretion of extracellular 36 enzymes should be responsible for a more pronounced decline in the S. sclerotiorum inoculum levels in soil (Woo et al., 2006). Among 20 strains of Trichoderma spp. evaluated for management of S. sclerotiorum in common beans, T. asperellum (cryptic sister species of T. asperelloides) exhibited the highest secretion of cell wall-degrading enzymes (CWDE) activity (Lopes et al., 2012). These data are consistent with those observed by Qualhato et al. (2013), where T. asperellum was effective against Fusarium solani, Rhizoctonia solani and S. sclerotiorum, and its antagonistic activity was associated with high activity of chitinase, β-1,3-glucanase and acid phosphatases. In our study, T. asperelloides CMAA 1584 displayed a great ability to inhibit myceliogenic germination and further degrade sclerotia of S. sclerotiorum by direct parasitism, outperforming T. lentiforme CMAA 1585 in this particular attribute. According to Geraldine et al. (2013), NAGase (N-β-acetylglucosaminidase) and β-1,3-glucanase enzymes play a key role in reducing the number of apothecia and the chain of events in the field that account for white mold severity, underlining the importance of these CWDEs in the control of white mold. On the other hand, T. lentiforme CMAA 1585 demonstrates to be more suitable as a biostimulant due to its ability to boost growth of cotton plants (Table 5, Figures 4 and 5). The high phosphate solubilization in the soil (Table 1) displayed by this strain and better development of cotton roots are possible mechanisms associated with plant growth enhancement. Many authors have detailed the ability of Trichoderma spp. to modulate physiological, biochemical, and molecular mechanisms in a wide assortment of plants under various growth conditions (Hermosa et al., 2013; Rubio et al., 2014, 2017; Elkelish et al., 2020), by the production phytohormones and a plethora of secondary metabolites (Jaroszuk-Ściseł et al., 2019). Using in vitro bioassay, Contreras-Cornejo et al. (2009) showed that T. virens Gv29.8 and T. atroviride IMI206040 can synthesize indole-acetic acid [IAA] (and some of its 37 derivatives), and suggests that the higher lateral root development observed in Arabidopsis wildtype plants is mediated by auxins. IAA synthesized by plant root-associated microorganisms can interfere with plant development by disturbing the auxin balance in plants, which can modify root architecture, increase root mass, and consequently, increase nutrient uptake by well-developed root system (Contreras-Cornejo et al., 2009). Sofo et al. (2011) reported that cherry rootstocks treated with T. harzianum commercial strain T-22 resulted in increased root and shoot growth by 76% and 61%, respectively. Furthermore, in mass spectrometry analyses these authors found that IAA and gibberellic acid (GA) levels were significantly increased by 40% and 143% in the roots, and by 49% and 71% in leaves, respectively. Gravel et al. (2007) suggested that growth promotion, in tomato seedling, is associated with the reduced ethylene (ET) production resulting from a decrease in its precursor 1- aminocyclopropane-1-carboxylic acid (ACC), and/or through the ACC deaminase (ACCD) activity present in the microorganism. Another possible mechanism arises from increased plant tolerance to abiotic stresses and/or by mitigation of damages caused by the accumulation of reactive oxygen species (ROS) in stressed plants (Mastouri et al., 2010). Thus, it can be hypothesized that the absence of significant results (P > 0.05) in seed germination and seedling vigor index in our assay under laboratory conditions, using the germitest paper method, the growth promotion may be related to the absence of environmental stresses. When evaluating the germination and vigor of wheat seedlings (Triticum aestivum L.) after seed treatment with different strains of Trichoderma spp., Anjum et al. (2020) obtained results that corroborate this scenario, because in a greenhouse trial, the positive outcomes were significantly more expressive than those observed in in vitro test conducted in the laboratory. In the present study, although the germination index was similar for these Trichoderma strains in both growth conditions, cotton plants from the mock control group exhibited less growth in the greenhouse 38 trial when compared with plants derived from the Trichoderma treatments, most likely due to the exposition of mock plants to suboptimal environmental conditions in contrast to higher resilience and improved growth promotion afforded by Trichoderma as a biological inoculant. Both Trichoderma strains may be further tested under field conditions, but with different purposes. As such, we propose that T. asperelloides CMAA 1584 should be designated to control sclerotia of S. sclerotiorum, while T. lentiforme CMAA 1585 would assume a role as a biostimuant due to its ability to promote better growth of cotton plants. Overall, these selected Trichoderma strains are suitable for application in consortium targeting both pathogen control and growth promotion in cotton crops with a consequent contribution to diminishing the reliance on chemical fertilizers and fungicides. Acknowledgements This study was supported by Empresa Brasileira de Pesquisa Agropecuária (Embrapa SEG 20.19.02.006.00.00), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. Wagner Bettiol (CNPq 307855/2019-8) acknowledges Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq for the productivity fellowship. This work was supported in part by the U.S. Department of Agriculture, Agricultural Research Service (Project Number: 5010-22410-024-00-D). Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. The mention of firm names or trade products does not imply they are endorsed or recommended by the USDA over other firms or similar products not mentioned. USDA is an equal opportunity provider and employer. Author contributions 39 WB, GMM, and LGS conceived and designed the laboratory and greenhouse experiments. LGS, RCC and PSON performed the laboratory and greenhouse experiments, and analyzed the data. WB, and GMM contributed with reagents/materials/analysis tools. CD provided critical analysis and editorial enhancements. All authors wrote the manuscript. All authors read and approved the final manuscript. Conflict of interest All authors declare that there is no conflict of interest in this original article. References Abdul‐Baki, A. A., and Anderson, J. D. (1973). Vigor determination in soybean seed by multiple criteria 1. Crop Sci. 13, 630-633. doi: 10.2135/cropsci1973.0011183X001300060013x Abdullah, M. T., Ali, N. Y., and Suleman, P. (2008). Biological control of Sclerotinia sclerotiorum (Lib.) de Bary with Trichoderma harzianum and Bacillus amyloliquefaciens. Crop Prot. 27, 1354-1359. doi: 10.1016/j.cropro.2008.05.007 ABRAPA - Associação Brasileira dos Produtores de Algodão (2021). Algodão no Brasil. Available at: https://www.abrapa.com.br/Paginas/Dados/Algodão no Brasil.aspx [Accessed February 13, 2021]. Anjum, Z. A., Hayat, S., Ghazanfar, M. U., Ahmad, S., Adnan, M., and Hussian, I. (2020). Does seed priming with Trichoderma isolates have any impact on germination and seedling vigor of wheat. Int. J. Botany Stud. 5, 65-68. Atanasova, L., Le Crom, S., Gruber, S., Coulpier, F., Seidl-Seiboth, V., Kubicek, C. P., et al. (2013). Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism. BMC Genomics 14, 121. doi: 10.1186/1471-2164-14-121 Barroso, P. A. V., Suassuna, N. D., Pedrosa, M. B., Morello, C. de L., Silva Filho, J. L. da, Lamas, F. M., et al. (2017). BRS 368RF: A glyphosate tolerant, midseason upland cotton cultivar for Northeast and North Brazilian cerrado. Crop Breed. Appl. Biotechnol. 17, 399- 402. doi: 10.1590/1984-70332017v17n4c59 Bell, D. K., Wells, W. D., and Markham, C. R. (1982). In vitro antagonism of Trichoderma species against six fungal plant pathogens. Phytopathology 72, 379. doi: 10.1094/Phyto- 72-379 Bettiol, W., de Medeiros, F. H. V., Barros Chiaramonte, J., and Mendes, R. (2021). “Advances 40 in screening approaches for the development of microbial bioprotectants to control plant diseases,” in Microbial bioprotectants for plant disease management, eds. J. Köhl and W. Ravensberg (London: Burleigh Dodds Science Publishing), 33-86. doi: 10.19103/AS.2021.0093.02 Boland, G. J., and Hall, R. (1994). Index of plant hosts of Sclerotinia sclerotiorum. Can. J. Plant Pathol. 16, 93-108. doi: 10.1080/07060669409500766 Brazil (2015). Portaria N 5, de 21 de agosto de 2015 - Diário Oficial da União no 161, Brasília, DF, 24 de julho de 2015. Available at: https://www.in.gov.br/web/guest/materia/- /asset_publisher/Kujrw0TZC2Mb/content/id/32414403/do1-2015-08-24-portaria-n-5-de- 21-de-agosto-de-2015-3241423 [Accessed February 15, 2021]. Brotman, Y., Lisec, J., Méret, M., Chet, I., Willmitzer, L., and Viterbo, A. (2012). Transcript and metabolite analysis of the Trichoderma-induced systemic resistance response to Pseudomonas syringae in Arabidopsis thaliana. Microbiology 158, 139146. doi: 10.1099/mic.0.052621-0 Charchar, M. J. D., Anjos, J. R. N. dos, and Ossipi, E. (1999). Ocorrência de nova doença do algodoeiro irrigado, no Brasil, causada por Sclerotinia sclerotiorum. Pesqui. Agropecu. Bras. 34, 1100-1106. doi: 10.1590/S0100-204X1999000600024 Chaverri, P., Branco-Rocha, F., Jaklitsch, W., Gazis, R., Degenkolb, T., and Samuels, G.J. (2015). Systematics of the Trichoderma harzianum species complex and the re- identification of commercial biocontrol strains. Mycologia 107, 558-590. doi: 10.3852/14- 147 CONAB - Companhia Nacional de Abastecimento (2022). Acompanhamento da safra brasileira de grãos - 11º levantamento. Available at: https://www.conab.gov.br/info- agro/safras/graos/boletim-da-safra-de-grao [Accessed August 13, 2022]. Contreras-Cornejo, H. A., Macías-Rodríguez, L., Cortés-Penagos, C., and López-Bucio, J. (2009). Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis. Plant Physiol. 149, 1579-1592. doi: 10.1104/pp.108.130369 Contreras-Cornejo, H. A., Macias-Rodriguez, L., Herrera-Estrella, A., and Lopez-Bucio, J. (2014). The 4-phosphopantetheinyl transferase of Trichoderma virens plays a role in plant protection against Botrytis cinerea through volatile organic compound emission. Plant Soil 379, 261-274. doi: 10.1007/s11104-014-2069-x Druzhinina, I. S., Seidl-Seiboth, V., Herrera-Estrella, A., Horwitz, B. A., Kenerley, C. M., Monte, E., et al. (2011). Trichoderma: the genomics of opportunistic success. Nat. Rev. Microbiol. 9, 749-759. doi: 10.1038/nrmicro2637 Elias, L. M., Domingues, M. V. P. F., Moura, K. E. de, Harakava, R., and Patricio, F. R. A. (2016). Selection of Trichoderma isolates for biological control of Sclerotinia minor and S. sclerotiorum in lettuce. Summa Phytopathol. 42, 216-221. doi: 10.1590/0100- 5405/2147 https://doi.org/10.3852/14-147 https://doi.org/10.3852/14-147 41 Elkelish, A. A., Alhaithloul, H. A. S., Qari, S. H., Soliman, M. H., and Hasanuzzaman, M. (2020). Pretreatment with Trichoderma harzianum alleviates waterlogging-induced growth alterations in tomato seedlings by modulating physiological, biochemical, and molecular mechanisms. Environ. Exp. Bot. 171, 103946. doi: 10.1016/j.envexpbot.2019.103946 Ferraz, L. de C. L., Nasser, L. C. B., and Café-Filho, A. C. (2011). Viabilidade de escleródios de Sclerotinia sclerotiorum e incidência de fungos antagonistas em solo de Cerrado. Summa Phytopathol. 37, 208-210. doi: 10.1590/S0100-54052011000400009 Garcia, R. A., Juliatti, F. C., and Cassemiro, T. A. (2012). Production of sclerotia on Sclerotinia sclerotiorum (Lib.) de Bary in culture media. Biosci. J. 28, 1-7. Geraldine, A. M., Lopes, F. A. C., Carvalho, D. D. C., Barbosa, E. T., Rodrigues, A. R., Brandão, R. S., et al. (2013). Cell wall-degrading enzymes and parasitism of sclerotia are key factors on field biocontrol of white mold by Trichoderma spp. Biol. Control 67, 308- 316. doi: 10.1016/j.biocontrol.2013.09.013 Görgen, C. A., Silveira Neto, A. N. da, Carneiro, L. C., Ragagnin, V., and Lobo Junior, M. (2009). Controle do mofo-branco com palhada e Trichoderma harzianum 1306 em soja. Pesqui. Agropecu. Bras. 44, 1583-1590. doi: 10.1590/S0100-204X2009001200004 Gravel, V., Antoun, H., and Tweddell, R. J. (2007). Growth stimulation and fruit yield improvement of greenhouse tomato plants by inoculation with Pseudomonas putida or Trichoderma atroviride: Possible role of indole acetic acid (IAA). Soil Biol. Biochem. 39, 1968-1977. doi: 10.1016/j.soilbio.2007.02.015 Grimes, D. W., and Carter, L. M. (1969). A linear rule for direct nondestructive leaf area measurements 1. Agron. J. 61, 477-479. doi: 10.2134/agronj1969.00021962006100030048x Haddad, P. E., Leite, L. G., Lucon, C. M. M., and Harakava, R. (2017). Selection of Trichoderma spp. strains for the control of Sclerotinia sclerotiorum in soybean. Pesqui. Agropecu. Bras. 52, 1140–1148. doi: 10.1590/s0100-204x2017001200002 Harman, G. E., Howell, C. R., Viterbo, A., Chet, I., and Lorito, M. (2004). Trichoderma species - opportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2, 43-56. doi: 10.1038/nrmicro797 Henis, Y., Adams, P. B., Lewis, J. A., and Papavizas, G. C. (1983). Penetration of sclerotia of Sclerotium rolfsii by Trichoderma spp. Phytopathology 73, 1043-1046. doi: 10.1094/Phyto-73-1043 Hermosa, R., Viterbo, A., Chet, I., and Monte, E. (2012). Plant-beneficial effects of Trichoderma and of its genes. Microbiology 158, 17-25. doi: 10.1099/mic.0.052274-0 Hermosa, R., Rubio, M. B., Cardoza, R. E., Nicolás, C., Monte, E., and Gutiérrez, S. (2013). The contribution of Trichoderma to balancing the costs of plant growth and defense. Internat. Microbiol. 16, 69-80. doi: 10.2436/20.1501.01.181 42 IMEA - Instituto Mato-Grossense de Economia Agropecuária (2019). Custos de Produção do algodoeiro no Mato Grosso. Available at: https://www.imea.com.br/imea-site/relatorios- mercado-detalhe?c=1&s=3 [Accessed January 2, 2020]. IMEA - Instituto Mato-Grossense de Economia Agropecuária (2021). 5a Estimativa da Safra 2020/21: Algodão. Available at: http://www.imea.com.br/ imea-site/relatorios-mercado- detalhe?c=1&s=9 [Accessed January 2, 2021]. Jaroszuk-Ściseł, J., Tyśkiewicz, R., Nowak, A., Ozimek, E., Majewska, M., Hanaka, A., et al. (2019). Phytohormones (auxin, gibberellin) and ACC deaminase in vitro synthesized by the mycoparasitic Trichoderma DEMTkZ3A0 strain and changes in the level of auxin and plant resistance markers in wheat seedlings inoculated with this strain conidia. Int. J. Mol. Sci. 20, 4923. doi: 10.3390/ijms20194923 Kniss, A. R. (2017). Long-term trends in the intensity and relative toxicity of herbicide use. Nat. Commun. 8, 14865. doi: 10.1038/ncomms14865 Köhl, J., Postma, J., Nicot, P., Ruoco, M., and Blum, B. (2011). Stepwise screening of microorganisms for commercial use in biological control of plant-pathogenic fungi and bacteria. Biol. Control 57, 1-12, doi:10.1016/j.biocontrol.2010.12.004 Komárek, M., Čadková, E., Chrastný, V., Bordas, F., and Bollinger, J.-C. (2010). Contamination of vineyard soils with fungicides: A review of environmental and toxicological aspects. Environ. Int. 36, 138-151. doi: 10.1016/j.envint.2009.10.005 Li, G. Q., Huang, H. C., Acharya, S. N., and Erickson, R. S. (2005). Effectiveness of Coniothyrium minitans and Trichoderma atroviride in suppression of Sclerotinia blossom blight of alfalfa. Plant Pathol. 54, 204-211. doi: 10.1111/j.1365-3059.2005.01119.x Li, N., Alfiky, A., Wang, W., Islam, M., Nourollahi, K., Liu, X., et al. (2018). Volatile compound-mediated recognition and inhibition between Trichoderma biocontrol agents and Fusarium oxysporum. Front. Microbiol. 9, 2614. doi: 10.3389/fmicb.2018.026 Liang, H.-J., Di, Y.-L., Li, J.-L., and Zhu, F.-X. (2015). Baseline sensitivity and control efficacy of fluazinam against Sclerotinia sclerotiorum. Eur. J. Plant Pathol. 142, 691-699. doi: 10.1007/s10658-015-0644-5 Lopes, F. A. C., Steindorff, A. S., Geraldine, A. M., Brandão, R. S., Monteiro, V. N., Lobo Júnior, M., et al. (2012). Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum. Fungal Biol. 116, 815-824. doi: 10.1016/j.funbio.2012.04.015 Lorito, M., Woo, S. L., Harman, G. E., and Monte, E. (2010). Translational research on Trichoderma : From ’omics to the field. Annu. Rev. Phytopathol. 48, 395–417. doi: 10.1146/annurev-phyto-073009-114314 Mao, X. W., Li, J. S., Chen, Y. L., Song, X. S., Duan, Y. B., Wang, J. X., et al. (2018). Resistance risk assessment for fluazinam in Sclerotinia sclerotiorum. Pestic. Biochem. Phys. 144, 27-35. doi: 10.1016/j.pestbp.2017.10.010 43 Mastouri, F., Björkman, T., and Harman, G. E. (2010). Seed treatment with Trichoderma harzianum alleviates biotic, abiotic, and physiological stresses in germinating seeds and seedlings. Phytopathology 100, 1213-1221. doi: 10.1094/PHYTO-03-10-0091 Meyer, M. C., Campos, H. D., Godoy, C. V., and Utiamada, C. M. (2014). Ensaios cooperativos de controle químico de mofo branco na cultura da soja: safras 2009 a 2012. Londrina Available at: https://ainfo.cnptia.embrapa.br/digital/bitstream/item/101371/1/Ensaios- cooperativos-de-controle-quimico-de-mofo-branco-na-cultura-da-soja-safras-2009-a- 2012.pdf. Meyer, M. C.; Campos, H. D.; Godoy, C. V.; Utiamada, C. M.; Sato, L. N.; Dias, A. R., et al. (2020). Eficiência de fungicidas para controle de mofo-branco (Sclerotinia sclerotiorum) em soja, na safra 2019/20: Resultados sumarizados dos ensaios cooperativos. Londrina Available at: http://ainfo.cnptia.embrapa.br/digital/bitstream/item/148978/1/CT-122.pdf. Monte, E., Bettiol, W., and Hermosa, R. (2019). “Trichoderma e seus mecanismos de ação para o controle de doenças de plantas,” in Trichoderma: Usos na agricultura, eds. M. C. Meyer, S. M. Mazaro, and J. C. da Silva (Brasília: Embrapa Soja), 181-200. Monte, E., and Hermosa, R. (2021). “The use of Trichoderma spp. to control plant diseases,” in Microbial bioprotectants for plant disease management, eds. J. Köhl and W. Ravensberg (London), 401-428. doi: 10.19103/AS.2021.0093.13 Morello, C. de L., Suassuna, N. D., Barroso, P. A. V., Filho, J. L. da S., Ferreira, A. C. de B., Lamas, F. M., et al. (2015). BRS 369RF and BRS 370RF: Glyphosate tolerant, high- yielding upland cotton cultivars for central Brazilian savanna. Crop Breed. Appl. Biotechnol. 15, 290-294. doi: 10.1590/1984-70332015v15n4c49 Murphy, J., and Riley, J. P. (1962). A modified single solution method for the determination of phosphate in natural waters. Anal. Chim. Acta 27, 31-36. doi: 10.1016/S0003- 2670(00)88444-5 Muthukumar, A., Eswaran, A., and Sanjeevkumas, K. (2011). Exploitation of Trichoderma species on the growth of Pythium aphanidermatum in chilli. Braz. J. Microbiol. 42, 1598- 1607. doi: 10.1590/S1517-83822011000400047 Napoleão, R., Nasser, L., Lopes, C., and Café Filho, A. (2006). Neon-S, novo meio para detecção de Sclerotinia sclerotiorum em sementes. Summa Phytopathol. 32, 180-182. doi: 10.1590/S0100-54052006000200014 Nautiyal, C. S. (1999). An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Lett. 170, 265-270. doi: 10.1111/j.1574- 6968.1999.tb13383.x O’Sullivan, C. A., Belt, K., and Thatcher, L. F. (2021). Tackling control of a cosmopolitan phytopathogen: Sclerotinia. Front. Plant Sci. 12, 1764. doi: 10.3389/fpls.2021.707509 Qualhato, T. F., Lopes, F. A. C., Steindorff, A. S., Brandão, R. S., Jesuino, R. S. A., and Ulhoa, C. J. (2013). Mycoparasitism studies of Trichoderma species against three 44 phytopathogenic fungi: evaluation of antagonism and hydrolytic enzyme production. Biotechnol. Let. 35, 1461-1468. doi: 10.1007/s10529-013-1225-3 Rubio, M. B., Quijada, N. M., Pérez, E., Domínguez, S., Monte, E., and Hermosa, R. (2014). Identifying beneficial qualities of Trichoderma parareesei for plants. Appl. Environ. Microbiol. 80, 1864-1873. doi: 10.1128/AEM.03375-13 Rubio, M. B., Hermosa, R., Vicente, R., Gómez-Acosta, F. A., Morcuende, R., Monte, E., and Bettiol, W. (2017). The combination of Trichoderma harzianum and chemical fertilization leads to the deregulation of pytohormone networking, preventing the adaptive responses of tomato plants to salt stress. Front. Plant Sci. 8, 294. doi: 10.3389/fpls.2017.00294 Samuels, G.J., Ismaiel, A., Bon, M.C., De Respinis. S., and Petrini, O. (2010). Trichoderma asperellum sensu lato consists of two cryptic species. Mycologia 102, 944-966. doi: 10.3852/09-243 Schwartz, H. F., and Singh, S. P. (2013). Breeding common bean for resistance to white mold: A Review. Crop Sci. 53, 1832-1844. doi: 10.2135/cropsci2013.02.0081 Sharma, P., and Sain, S. K. (2010). Induction of systemic resistance in tomato and cauliflower by Trichoderma spp. against stalk rot pathogen, Sclerotinia sclerotiorum (Lib) de Bary. J. Biol. Control 18, 21-28. doi: 10.18311/jbc/2004/4042 Siddiquee, S., Cheong, B. E., Taslima, K., Kausar, H., and Hasan, M. M. (2012). Separation and identification of volatile compounds from liquid cultures of Trichoderma harzianum by GC-MS using three different capillary columns. J. Chromatogr. Sci. 50, 358-367. doi: 10.1093/chromsci/bms012 Silva Neto, S. P. da, Pereira, A. F., Morello, C. de L., and Suassuna, N. D. (2016). “Melhoramento clássico e biotecnologia visando à superação de desafios,” in Desafios do Cerrado: Como sustentar a expansão da produção com produtividade e competitividade. Associação Mato-grossense dos Produtores de Algodão, eds. G. V. Piccoli, D. Endrigo, and A. L. Maurício (Cuiabá: Associação mato-grossense dos produtores de algodão), 215- 252. Silva, J. C., Bettiol, W., and Suassuna, N. D. (2019). Ramularia leaf spot: an emergent disease of cotton in Brazil. Trop. Plant Pathol. 44, 473-482. doi: 10.1007/s40858-019-00308-w Sivakumar, S., Prem Kumar, G., Vinoth, S., Siva, G., Vigneswaran, M., Gurusaravanan, P., et al. (2021). Temporal expression profiling of GhNAC transcription factor genes in cotton cultivars under abiotic stresses. Plant Gene 28, 100334. doi: 10.1016/j.plgene.2021.100334 Smolińska, U., and Kowalska, B. (2018). Biological control of the soil-borne fungal pathogen Sclerotinia sclerotiorum - a review. J. Plant Pathol. 100, 1-12. doi: 10.1007/s42161-018- 0023-0 Sofo, A., Scopa, A., Manfra, M., De Nisco, M., Tenore, G., Troisi, J., et al. (2011). Trichoderma harzianum strain T-22 induces changes in phytohormone levels in cherry rootstocks (Prunus cerasus × P. canescens). Plant Growth Regul. 65, 421-425. doi: 10.1007/s10725- 45 011-9610-1 Sood, M., Kapoor, D., Kumar, V., Sheteiwy, M. S., Ramakrishnan, M., Landi, M., et al. (2020). Trichoderma: The “secrets” of a multitalented biocontrol agent. Plants 9, 762. doi: 10.3390/plants9060762 Suassuna, N. D., Silva, J. C., and Bettiol, W. (2019). “Uso do Trichoderma na cultrua do algodão,” in Trichoderma: Usos na agricultura, eds. M. C. Meyer, S. M. Mazaro, and J. C. da Silva (Brasília: Embrapa Soja), 361-379. Sumida, C. H., Daniel, J. F. S., Araujod, A. P. C. S., Peitl, D. C., Abreu, L. M., Dekker, R. F. H., et al. (2018). Trichoderma asperelloides antagonism to nine Sclerotinia sclerotiorum strains and biological control of white mold disease in soybean plants. Biocontrol Sci. Technol. 28, 142-156. Tarazi, R., Jimenez, J. L. S., and Vaslin, M. F. S. (2019). Biotechnological solutions for major cotton (Gossypium hirsutum) pathogens and pests. Biotechnol. Res. Innovation 3, 19-26. doi: 10.1016/j.biori.2020.01.001 Tourneau, D. Le (1979). Morphology, cytology, and physiology of Sclerotinia species in culture. Phytopathology 69, 887-890. doi: 10.1094/Phyto-69-887 Troian, R. F., Steindorff, A. S., Ramada, M. H. S., Arruda, W., and Ulhoa, C. J. (2014). Mycoparasitism studies of Trichoderma harzianum against Sclerotinia sclerotiorum: evaluation of antagonism and expression of cell wall-degrading enzymes genes. Biotechnol. Lett. 36, 2095-2101. doi: 10.1007/s10529-014-1583-5 Verma, M., Brar, S. K., Tyagi, R. D., Surampalli, R. Y., and Valéro, J. R. (2007). Antagonistic fungi, Trichoderma spp.: Panoply of biological control. Biochem. Eng. J. 37, 1-20. doi: 10.1016/j.bej.2007.05.012 Woo, S. L., Scala, F., Ruocco, M., and Lorito, M. (2006). The molecular biology of the interactions between Trichoderma spp., phytopathogenic fungi, and plants. Phytopathology 96, 181-185. doi: 10.1094/PHYTO-96-0181 Wu, N., Yang, J., Wang, G., Ke, H., Zhang, Y., Liu, Z., et al. (2022). Novel insights into water- deficit-responsive mRNAs and lncRNAs during fiber development in Gossypium hirsutum. BMC Plant Biol. 22, 1-16. doi: 10.1186/s12870-021-03382-y Zhou, F., Zhang, X. L., Li, J. L., and Zhu, F. X. (2014). Dimethachlon resistance in Sclerotinia sclerotiorum in China. Plant Dis. 98, 1221-1226. doi: 10.1094/PDIS-10-13-1072-RE 46 Table 1. Phosphate solubilization by Trichoderma asperelloides CMAA 1584 and Trichoderma lentiforme CMAA 1585. Strain Phosphate solubilization (%) CMAA 1584 5.2 ± 0.79 b CMAA 1585 31.7 ± 4.08 a *Values represent means (± standard error) and when followed by the same letter do not differ significantly (Tukey’s test at p < 0.05). Table 2. Antagonistic activity of Trichoderma asperelloides CMAA 1584 and Trichoderma lentiforme CMAA 1585 against Sclerotinia sclerotiorum by dual culture test and production of volatile organic compounds (VOCs). Treatments Volatile organic compounds Dual culture Growth rate (mm day-1) Inhibition (%) Growth rate (mm day-1) Inhibition (%) Number of sclerotia CMAA 1584 23.6 ± 1.2 b (53%) 41.9 ± 2.7 a 27.4 ± 0.1 a (10%) 9.5 ± 0.4 a 0.6 ± 0.6 a CMAA 1585 22.8 ± 1.5 b (55%) 43.8 ± 3.3 a 26.5 ± 0.6 a (13%) 12.2 ± 1.8 a 7.4 ± 1.5 b Control 42.5 ± 0.0 a - 30.5 ± 0.5 b - 15.6 ± 1.6 c *Values in each column represent means (± standard error) and when followed by the same letter do not differ significantly from each other (Tukey p < 0.05). Values between parentheses indicate the inhibition growth rate when compared to control. Table 3. Effect of Trichoderma asperelloides CMAA 1584 and Trichoderma lentiforme CMAA 1585 in cotton seeds germination and vigor index. Treatments (conidia mL-1) T. asperelloides CMAA 1584 T. lentiforme CMAA 1585 Germination (%) Vigor index1 Germination (%) Vigor index1 Control 92.5 ± 1.4 a 163.4 ± 1.5 a 92.5 ± 1.4 a 163.4 ± 1.5 a 1 × 106 91.2 ± 2.4 a 165.6 ± 4.6 a 92.5 ± 1.4 a 164.2 ± 3.6 a 1 × 107 88.7 ± 2.4 a 155.5 ± 8.3 a 92.5 ± 2.5 a 162.0 ± 3.8 a 1 × 108 93.7 ± 3.1 a 163.6 ± 6.1 a 91.2 ± 1.2 a 164.7 ± 2.3 a *Values in each column represent means (± standard error) and when followed by the same letter do not differ significantly (Tukey’s test at p < 0.05). 1Calculated according to Abdul-Baki and Anderson (1973) by the equation: Vigor Index = Germination (%) × Seedling Dry Weight (g). 47 Table 4. Leaf area, stem diameter, root length, plant height and fresh and dry weight of the root and aboveground of cotton plants treated with different concentrations of Trichoderma asperelloides CMAA 1584. Treatment Leaf area (cm2) Stem diameter (mm) Root fresh weight (g) Root dry weight (g) Aboveground fresh weight (g) Aboveground dry weight (g) Root length (cm) Plant height (cm) 60 DAS 55 DAS Control 22.6 ± 3.7 a 4.2 ± 0.4 a 16.2 ± 0.9 a 8.9 ± 0.7 a 16.0 ± 1.8 a 11.5 ± 1.0 a 107.4 ± 2.2 a 27.8 ± 3.1 a 1 × 106 25.7 ± 2.6 a 4.5 ± 0.2 a 16.9 ± 1.0 a 9.2 ± 0.4 a 15.4 ± 1.8 a 11.6 ± 0.6 a 108.4 ± 4.7 a 32.8 ± 1.5 a 1 × 107 44.9 ± 2.0 b 5.0 ± 0.3 a 19.8 ± 1.0 a 10.4 ± 0.7 a 19.4 ± 1.9 a 13.1 ± 1.2 a 105.8 ± 0.8 a 33.3 ± 2.9 a 1 × 108 32.0 ± 5.0 ab 5.0 ± 0.2 a 19.2 ± 1.4 a 9.5 ± 0.2 a 19.8 ± 2.0 a 12.9 ± 0.6 a 105.2 ± 2.5 a 34.2 ± 1.3 a CV (%) 24.6 14.0 14.0 13.1 26.2 17.5 6.4 16.7 *Values in each column represent means (± standard error) and when followed by the same letter do not differ significantly (Tukey’s test at p < 0.05). DAS = day after sowing. Table 5. Leaf area, stem diameter, root length, height and fresh and dry weight of the root and aboveground of cotton plants treated with different concentrations of Trichoderma lentiforme CMAA 1585. Treatment Leaf area (cm2) Stem diameter (mm) Root fresh weight (g) Root dry weight (g) Aboveground fresh weight (g) Aboveground dry weight (g) Root length (cm) Plant height (cm) 60 DAS 55 DAS Control 22.6 ± 3.7 a 4.2 ± 0.4 a 16.2 ± 0.9 a 8.9 ± 0.7 a 16.0 ± 1.8 a 11.5 ± 1.0 a 107.4 ± 2.2 a 27.8 ± 3.1 a 1 × 106 26.1 ± 1.8 a 4.5 ± 0.1 ab 19.4 ± 2.3 a 9.9 ± 0.6 a 16.5 ± 0.7 a 12.7 ± 0.6 a 108.2 ± 3.8 a 33.8 ± 2.1 ab 1 × 107 26.4 ± 2.8 a 4.4 ± 0.1 ab 17.5 ± 0.2 a 8.6 ± 0.3 a 15.4 ± 0.4 a 10.8 ± 0.4 a 112.0 ± 2.4 a 30.0 ± 1.2 ab 1 × 108 33.7 ± 2.9 a 5.2 ± 0.3 b 30.3 ± 2.1 b 11.6 ± 0.3 b 27.1 ± 3.3 b 16.8 ± 1.1 b 110.2 ± 3.9 a 37.7 ± 2.4 b CV (%) 24.9 11.2 18.7 8.5 22.2 14.0 7.3 14.6 *Values in each column represent means (± standard error) and when followed by the same letter do not differ significantly (Tukey’s test at p < 0.05). DAS = day after sowing. 48 Figure 1. Morphological characterization of indigenous Trichoderma spp. (A) Five-day-old PDA-grown cultures of Trichoderma asperelloides (CMAA 1584); (B) Microscopic images showing conidiophores of CMAA 1584 (magnification at 200 ×); (C) Typical conidia of CMAA 1584 (magnification at 200 ×); (D) Five-day-old PDA-grown cultures of Trichoderma lentiforme (CMAA 1585); (E) Microscopic images showing conidiophores of CMAA 1585 (magnification at 200 ×); (F) Typical conidia of CMAA 1585 (magnification at 200 ×). Figure 2. Biocontrol potential of Trichoderma strains against Sclerotinia sclerotiorum according to Bell scale. Bars indicate mean ± standard error. 49 Figure 3. Ability of Trichoderma asperelloides CMAA 1584 and Trichoderma lentiforme CMAA 1585 strains to inhibit myceliogenic germination (A) and to parasitize (B) Sclerotinia sclerotiorum sclerotia by direct antagonism at different inoculum concentrations. *Black diamond symbol represents means (± standard error) and the dashed line represents the fitted quadratic curve. 50 Figure 4. Cotton plants treated with Trichoderma asperelloides CMAA 1584 (A, B) and Trichoderma lentiforme CMAA 1585 (C, D) after 60 days of sowing in rhizotron. 51 Figure 5. Fresh and dry weight of the root (A and B) and aboveground (C and D), stem diameter (E), and plant height (F) of cotton cultivar FM 975 WS® with Trichoderma lentiforme CMAA 1585. Bars indicate standard error. 52 CHAPTER 2 BIOREACTOR-IN-A-GRANULE DESIGNED FOR Trichoderma asperelloides USING RICE FLOUR AND ITS EFFICACY AGAINST Sclerotinia sclerotiorum Lucas Guedes Silva1,2, Renato Cintra Camargo2, Gabriel Moura Mascarin2, Camila Patrícia Favaro3,4, Cristiane Sanchez Farinas4, Caue Ribeiro de Oliveira4, Wagner Bettiol2* 1 Department of Plant Protection, School of Agriculture, São Paulo State University, 18610- 034, Botucatu, SP, Brazil 2 Embrapa Environment, SP 340 Road, Km 127.5, 13918-110, Jaguariúna, SP, Brazil 3 Department of Chemical Engineering, Federal University of São Carlos, 13565-905, São Carlos, SP, Brazil 4 Embrapa Instrumentation, November XV, nº 1.452, 13560-970, São Carlos, SP, Brazil *Corresponding author: Wagner Bettiol - wagner.bettiol@embrapa.br 53 Abstract The replacement of cereal grains by agro-industrial by-products (wastes) for mass production of biological control agents (BCA) allied to the development of stable formulations with extended shelf-life represents a critical step for advancing the use of BCA. This study aimed to optimize the Trichoderma asperelloides stationary fermentation in rice flour, an inexpensive and abundant residue in Brazil, to sustain high yields of conidial production and to subsequently develop a rice flour-based formulation designed to simulate a microbioreactor that affords in- situ conidiation, extended shelf-life, and effective control of Sclerotinia sclerotiorum, the most devastating fungal pathogen of annual legume crops. The conidial yield of T. asperelloides in rice flour was mainly influenced by nitrogen content (0.1% w/w) and by the fermentor type (Erlenmeyer flask). Hydrolyzed yeast (Hilyses®) was the best source of nitrogen combined with rice flour with a resultant yield of 2.6 × 109 CFU g-1 within 14 days. Subsequently, five formulations (GControl, GLecithin, GBreak-Thru, GBentonite and GOrganic compost + Break-Thru) were obtained by extrusion and assessed for their potential to induce secondary sporulation in situ, storage stability, and efficacy against S. sclerotiorum. Formulations GControl, GBreak-Thru, GBentonite and GOrganic compost + Break-Thru stood out with the highest number of CFU after sporulation upon re- hydration on water-agar medium. The shelf-life at room temperature (~25 °C) was maintained invariably for up to 3 months with formulations GControl and GBentonite, while the fungus remained viable for 12 months with GBentonite and GOrganic + Break-Thru formulations during refrigerated storage (4 °C). Formulations exhibited similar efficacy in suppressing the myceliogenic germination of S. sclerotiorum irrespective of application rates (i.e., 5 × 104, 5 × 105 or 5 × 106 CFU g-1 of soil), resulting in 79.2, 87.5 and 93.7% of sclerotial inhibition, respectively. Significant number of degraded and dead sclerotia was observed with increased dose of GControl formulation, ranging from 2.1 to 23.0%. Keywords: Bioprotectant, extrusion, solid-state fermentation, white mold, shelf-life. 54 INTRODUCTION Trichoderma is a well-known genus for its multi-beneficial roles in agriculture, including its wide biocontrol activity against several plant pathogens, plant growth promotion, and mitigation of abiotic stresses in plants (Hermosa et al., 2012; Lorito et al., 2010; Morán- Diez et al., 2020; Rubio et al., 2017). Trichoderma-based products, such as bioprotectors and biofertilizers, play pivotal role in food production worldwide as they can potentially minimize the chemical fertilizer inputs in agricultural crops (Bettiol et al., 2019; Harman et al., 2010; Woo et al., 2014). According to Bueno et al. (2020), Trichoderma was applied in more than 5.5 million hectares (ha) of soybean in Brazil in 2017 for the management of soilborne diseases. This area has increased in recent years, possibly reaching 20 million ha, and it is by far the most sold fungal biofungicide in Brazil due to its easiness in mass production allied to its multiple properties in plant protection and health. Among the soilborne plant pathogens that affect soybean growth and yield, white mold, also known as Sclerotinia stem rot, caused by the ascomycete fungus Sclerotinia sclerotiorum (Lib.) de Bary, is one of the most globally destructive diseases (Boland; Hall, 1994). According to Meyer et al. (2019), S. sclerotiorum is endemic in approximately 27% of soybean production areas in Brazil, and its damages can result in economic losses of up to US$ 1.47 billion annually (Lehner et al., 2017). This fungus infects leaves, flowers, stems, and pods of the host plants, forming resistant structures, known as sclerotia, which are able to survive in soil or on crop debris for several years (Bolton et al., 2006). Thus, the management of S. sclerotiorum occurs at several stages of crop development and usually requires integration of multiple methods, including biological control specifically directed to targeting the pathogen’s sclerotia remained in soil (Smolińska; Kowalska, 2018). The bioproduct manufacturers can use three different fermentation strategies in order to mass produce Trichoderma spp.: solid, liquid, and biphasic fermentation (Jin; Custis, 2011; 55 Mascarin et al., 2019). Currently, manufacturers employ mostly biphasic fermentation systems, in which the inoculum is initially produced by liquid culture media and subsequently transferred to solid substrates (rice, barley, wheat, oat, or millet) for induction of aerial conidiation or simply sporulation that leads to a sheer number of conidia, the main active ingredient for different types of formulation (Li et al., 2010; Jin; Custis, 2011; Mascarin et al., 2010, 2019; Woo et al., 2014). In this system, harvesting the spores is mandatory to concentrate the fungal biomass for the formulation process (Faria; Wraight, 2007; Li et al., 2010; Mascarin et al., 2019). However, throughout this process, metabolites which harbor antimicrobial properties and/or act as plant stimulants are inevitably lost. Additionally, the solid residue after spore extraction needs proper destination, which may be explored in energy production (Elias et al., 2022) or composting. Growth substrate is the bottleneck for the production of cost-effective and quality BCAs, representing up to 50% of production costs (Eltem et al., 2014; Stanbury et al., 2017). Thus, the recent increases in paddy rice cost in Brazil, which reached more than 90% between January and December 2020 (CEPEA, 2021), directly impacts the production of fungal biocontrol agents that rely on this cereal grain for their growth. The use of agricultural by-products (wastes) for the production of fungal bioagents by the biopesticide industry offers a valuable alternative to the use of rice grains, as it allows the reduction of production costs and a more noble destination of this residue, especially in Brazil where these agricultural by-products are plenty and inexpensive (Farinas, 2015; Soccol et al., 2017). Another concern that arises from using rice as fungal growth substrate resides in its social importance as staple food and main source of energy for more than half of the world’s population, especially in Asian countries (Bird et al., 2000). Before reaching the consumer, several processing steps are carried out with the grain, which eventually generate several by- products (Esa et al., 2016). Broken rice, one of these by-products, represents between 10 to 15 56 % of all rice processed. Due to its low cost (about 1/3 to 1/2 of the brown rice), high availability, and good nutritional value (74% starch and 7% protein) (Liu et al., 2016), several technologies have been proposed to increase its use in the industry (Ahmed et al., 2015; Myburgh et al., 2019; Nakano et al., 2012), including in biological control manufacturers (Jaronski 2014; Bich et al., 2018). On another important aspect that must be taken into account in the production pipeline of microbial biopesticides concerns the development of waste-free and low-cost formulations that enable prolonged shelf-life, improved efficiency, and easy application of microbial biocontrol agents. Of particular interest, starchy compounds have successfully been used as a carrier or additive in formulations of plant beneficial microorganisms (Vassilev et al., 2020). In the bioencapsulation matrix, starch reduces the physical stress to microbial cells and significantly improves their survival (Bashan et al., 2002), as it provides structural support and protection against thermal, oxidative, and osmotic stresses (Chan et al., 2011; Schoebitz et al., 2012; Tal et al., 1999). Moreover, considering that Trichoderma has the ability to hydrolyze starch into simple sugars (Schellart et al., 1976; Asis et al., 2021), the use of starchy products for mass production and as a carrier in Trichod