Universidade Federal De Goiás Doutorado Em Biotecnologia E Biodiversidade Programa De Pós-Graduação Da Rede Pró Centro- Oeste

UNIVERSIDADE FEDERAL DE GOIÁS DOUTORADO EM BIOTECNOLOGIA E BIODIVERSIDADE PROGRAMA DE PÓS-GRADUAÇÃO DA REDE PRÓ CENTRO- OESTE

BIOPROSPECÇÃO DE BACTÉRIAS A PARTIR DA COMUNIDADE ENDOFÍTICA RADICULAR CULTIVÁVEL DE Aloe vera (L.) BURM. F. (Xanthorrhoeaceae)

Cintia Faria da Silva Doctor Scientiae

RIO VERDE GOIÁS – BRASIL 2019

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Nome completo do autor: Cintia Faria da Silva

Título do trabalho: Bioprospecção de bactérias à partir da comunidade endofítica radicular cultivável de Aloe vera (L.) Burm. F. (Xanthorrhoeaceae)

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1 Neste caso o documento será embargado por até um ano a partir da data de defesa. A extensão deste prazo suscita justificativa junto à coordenação do curso. Os dados do documento não serão disponibilizados durante o período de embargo. Casos de embargo: - Solicitação de registro de patente; - Submissão de artigo em revista científica; - Publicação como capítulo de livro; - Publicação da dissertação/tese em livro. 2 A assinatura deve ser escaneada. Versão atualizada em setembro de 2017. CINTIA FARIA DA SILVA

BIOPROSPECÇÃO DE BACTÉRIAS A PARTIR DA COMUNIDADE ENDOFÍTICA RADICULAR CULTIVÁVEL DE Aloe vera (L.) BURM. F. (Xanthorrhoeaceae)

Orientador:

Prof. Edson Luiz Souchie, PhD Co-orientador:

Prof. Marcos Antônio Soares, PhD

Tese apresentada à Universidade Federal de Goiás como parte das exigências do Programa de Pós-Graduação em Biotecnologia e Biodiversidade, para obtenção do título de Doctor Scientiae.

Rio Verde Goiás – Brasil 2019 Ficha de identificação da obra elaborada pelo autor, através do Programa de Geração Automática do Sistema de Bibliotecas da UFG.

Faria da Silva, Cintia Bioprospecção de bactérias à partir da comunidade endofítica radicular cultivável de Aloe vera (L.) Burm. F. (Xanthorrhoeaceae) [manuscrito] / Cintia Faria da Silva. - 2019. xi, 104 f.: il.

Orientador: Prof. Dr. Edson Luiz Souchie; co-orientador Dr. Marcos Antônio Soares. Tese (Doutorado) - Universidade Federal de Goiás, Instituto de Patologia Tropical e Saúde Pública (IPTSP), Programa de Pós graduação em Biotecnologia e Biodiversidade, Cidade de Goiás, 2019. Bibliografia. Inclui lista de figuras, lista de tabelas.

1. Babosa. 2. Micro-organismos. 3. Traços funcionais. 4. Promoção de crescimento vegetal. 5. Biotecnologia. I. Luiz Souchie, Edson, orient. II. Título.

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AGRADECIMENTOS

A Deus, por me amparar nos momentos difíceis e refúgio nas horas de cansaço. A minha família, meus pais, Severino e Maria Aparecida, e minhas irmãs Lessi, Silvia e Nádia, pelo apoio, incentivo e paciência no decorrer desta jornada. Aos meus sobrinhos Ana Lara, Humberto e Catarina. Ao professor Dr. Edson Luiz Souchie, pela orientação, amizade, confiança e incentivo, com palavras de apoio, acreditando na minha capacidade, fazendo com que eu sempre seguisse em frente. Ao professor Dr. Marcos Antônio Soares, pela disponibilidade em minha co- orientação. As professoras Dras. Maria Andréia Corrêa Mendonça e Luciana Cristina Vitorino por toda dedicação, ensinamentos e contribuições para o meu crescimento profissional. A professora Dra. Paula Fabiane Martins, que pelo pouco tempo em que passou em Rio Verde, pode dividir seu conhecimento e paixão pela microbiologia. Aos professores Dr. Paulo Sérgio Pereira e Dra. Erika Crispim Resende pela ajuda na parte química do trabalho. Ao professor da Universidade de São Paulo, Dr. Welington Luiz Araujo e a pós doutoranda, Dra. Manuella Nóbrega Dourado Ribeiro, por me receberem de portas abertas no Laboratório de Biologia Molecular e Ecologia Microbiana para contribuição em parte das análises moleculares. A minha amiga Andréia, que conheci no início da seleção do doutorado e dividiu comigo diversos momentos, em disciplinas em outras cidades e diversas horas de conversas por telefone, meu imenso carinho. A Paula que esteve comigo desde a época do mestrado, com carinho e companheirismo e amor a microbiologia, foi parceira de viagens, disciplinas e experimentos, participando do meu aprendizado e crescimento. Aos colegas e amigos que conquistei ao longo do mestrado e doutorado, especialmente ao Thiago, Deborah, Priscila, Marília, Kelly e Paula pelo convívio, aprendizado e momentos divididos juntos, sejam eles bons ou ruins. Às colegas e amigas Ana Paula, Carine, Michelle, Daniele, Mariluza, Luciana, Elisvane, Leticia, Vanessa e Ana Cláudia, que deixaram vários momentos mais suaves, com palavras de apoio e risadas.

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Aos companheiros do Laboratório de Microbiologia Agrícola que passaram por lá e aos que chegaram a pouco tempo, Isabel, Moacir, Tenille, Juliana, Lorena Lara, Elizabeth, Tonhão, Thales, Yasmin, Matheus, Natasha, Tâmara, Leônidas, Bárbara, Ana Flávia, Kelly, Fellipe e Lidiane, pela colaboração e convívio. Em especial ao alunos de inciação científica que participaram diretamente deste trabalho Heitor, Lázara, Milene, Leonardo, Thayná e Isabelle, que me ajudaram nas diversas etapas do trabalho. Muito obrigada! Ao Programa de Pós-graduação em Biotecnologia e Biodiversidade da Rede Pró Centro - Oeste, juntamente com a Universidade Federal de Goiás e o Instituto Federal Goiano pela oportunidade de realização deste trabalho. Ao Instituto Federal Goiano, campi Rio Verde e Iporá pela estrutura disponibilizada para execução do projeto. Às agências de fomento CAPES, CNPq e FAPEG, pelo apoio financeiro. A todos que direta ou indiretamente colaboraram para a realização deste trabalho o meu sincero agradecimento!

“A quem te pedir um peixe, dá uma vara de pescar.” Pensando bem, não só a vara de pescar, também a linhada, o anzol, a chumbada, a isca…”

Cora Coralina

BIOGRAFIA DA AUTORA

Cintia Faria da Silva, natural de Rio Verde – GO, filha de Severino Ferreira da Silva e Maria Aparecida Faria da Silva, graduada em Ciências – Licenciatura Plena Habilitação em Biologia pela Universidade de Rio Verde (2005), especialista em Microbiologia e Higiene de Alimentos pela Pontifícia Universidade Católica de Goiás (2009), mestra em Ciências Agrárias - Agronomia (2014) pelo Instituto Federal Goiano – Campus Rio Verde. Em agosto de 2014, ingressou no Programa de Pós-Graduação em Biotecnologia e Biodiversidade (Doutorado) da Rede Pró Centro-Oeste pela Universidade Federal de Goiás e Instituto Federal Goiano – Campus Rio Verde, concluindo em setembro de 2019.

SUMÁRIO

INDICE DE TABELAS ...... viii ÍNDICE DE FIGURAS ...... ix RESUMO ...... 1 2. REVISÃO DE LITERATURA ...... 5 2.1. Aloe vera ...... 5 2.2. Aloína ...... 6 2.4. Micro-organismos endofíticos ...... 8 2.5. Traços funcionais ...... 11 2.5.1. Síntese de fitormônios ...... 12 2.5.2. Solubilização de fosfatos ...... 13 2.5.3. Produção de sideróforos ...... 14 2.5.4. Controle biológico - Antibiose...... 15 3. REFERÊNCIAS BIBLIOGRÁFICAS ...... 17 3. JUSTIFICATIVA ...... 31 4. REFERÊNCIAS BIBLIOGRÁFICAS ...... 32 OBJETIVO GERAL ...... 34 OBJETIVOS ESPECÍFICOS ...... 35 CAPÍTULO I ...... 36 Artigo - Screening of plant growth-promoting endophytic bacteria from the roots of the medicinal plant Aloe vera ...... 37 CAPITULO II ...... 80 Artigo - Endophytic bacteria promote growth and increase the aloin content of Aloe vera ...... 81 CONCLUSÃO GERAL ...... 104

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INDICE DE TABELAS

Página CAPÍTULO I Table 1. In vitro production of indoleacetic acid (IAA) by bacteria obtained from the roots of Aloe vera.……………...... 49 Table 2. pH and in vitro solubilization of Bayóvar natural phosphate (P), tricalcium phosphate (Ca3(PO4)2), and iron phosphate (FePO4) by A. vera root endophytic bacteria …………………...... 51 Table 3. Inhibition of mycelial growth of phytopathogenic fungi Sclerotinia sclerotiorum, Fusarium sp., and Rhizoctonia sp. by endophytic bacteria in antagonism tests in vitro………………………………...... 54

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ÍNDICE DE FIGURAS

Página REVISÃO DE LITERATURA Figura 1. Tipos de endófitos e seu processo de colonização radicular (Hardoim et al., 2008) ...... 10

CAPÍTULO I Fig. 1. Environments from which Aloe vera roots were collected. A) Field: Farm; B) Garden of the Planta e Vida Cooperative; C) Nursery of the University of Rio Verde, GO; D) Extensive root-system of A. vera...... 41 Fig. 2. Percentage of endophytic bacteria isolated from the roots of A. vera. Bacteria were selected based on the production of indoleacetic acid and arbuscular microrrheic fungi (AMF) spores on the rhizospheric soil of the samples field, garden, and nursery. ( ) number of bacteria and number of spores.………………………………...... 47 Fig. 3. Similarity tree based on 16S gene sequences from endophytic bacteria isolated from the roots of A. vera in three different sites with relationships between the nearest strains. (T) Type strain. Letters following the isolate numbers indicate the sampling site (V = nursery, H = garden, and C = field). Black numbers below the nodes indicate the posterior probability, and the blue values above the nodes represent the bootstrap values based on 10,000 replicates...... 48 Fig. 4. Double culture test between root endophytic bacteria of A. vera and phytopathogens Sclerotinia sclerotiorum, Fusarium sp., and Rhizoctonia sp. (A) S. sclerotiorum (control); (B) Antagonism between the phytopathogenic fungus S. sclerotiorum and the bacterium 14V Enterobacter tabaci. (C) Fusarium sp. (control); (D) Antagonism between Fusarium sp. and the bacterium 149H Paraburkholderia sp. (E) Rhizoctonia sp. (control); (F) Antagonism between Rhizoctonia sp. and the bacterium 348V Enterobacter

ix tabaci...... 52 Fig. 5. Morphological alterations of the mycelium of the phytopathogenic fungus S. sclerotiorum are indicated by red arrows following interaction with root endophytic bacteria of A. vera. (A) Control - hyphae of S. sclerotiorum; (B) interaction with the 3V B. agri bacterium – hyphae swollen; (C) 14V Enterobacter tabaci bacterium - hyphae twisted; (D) bacterium 17V E. tabaci - degenerative changes in the morphology of hyphae; (E) 383H L. macroides bacteria – hyphae collapsed or swollen………………………………………... 53 Fig. 6. Multifunctional potential of endophytic bacteria associated with the roots of A. vera from different sites. A) Number of bacterial isolates expressing potential functional traits for biotechnology. B) Number of isolates presenting each functional trait evaluated (IAA biosynthesis, amylase production, Bayóvar phosphate solubilization, calcium and iron, and antibiosis to phytopathogens S. sclerotiorum, Fusarium sp., and Rhizoctonia sp.)…………………………………………………………………………...... 55 Fig. 7. Endophytic bacteria 149H Paraburkolderia sp., and 135V and 348V Enterobacter tabaci isolates from the roots of A. vera and functional traits expressed by these isolates. Arrows indicate that the isolate expresses the indicated trait. …………………………………………………………………. 56

CAPÍTULO II Fig. 1. Chromatogram of the standard aloin, extracted from Aloe vera, with 125 μg.mL-1 and the UV absorption spectrum...... 87 Fig. 2. Biometric parameters of the Aloe vera plants inoculated with different strains of endophytic bacteria. Uppercase letters compare with the Control treatment and lowercase letters compared with the MIX treatment. (A) Length (cm) of the shoot plant; (B) Number of leaves; (C) Fresh weight (g) of the leaves and root; (D) Dry weight (g) of the leaves and root; (E) Root volume (mm3); (F) Surface area (mm2) of the root, and (G) Diameter of the root (mm). Control = not inoculated; 35V = Enterobacter ludwigii; 135V = Enterobacter tabaci; 149H = Paraburkholderia sp.; 389C = Pantoea cypripedii; MIX = consortium of the four bacteria. In each graph, pairs of columns with different letters at the top have significantly different means (Dunnett) (mean of four

x replicates)…………………………………………………………..………………………………………….. 89 Fig. 3. Aloin content of Aloe vera plants inoculated with different strains of endophytic bacteria: (A) Dry gel; B) Dry latex. Control = not inoculated; 35V = Enterobacter ludwigii; 135V = Enterobacter tabaci; 149H = Paraburkholderia sp.; 389C = Pantoea cypripedii; MIX = consortium of the four bacteria. In each graph, pairs of columns with different letters at the top have significantly different means (Tukey, 5%)……………………………………………… 90

RESUMO

SILVA, Cintia Faria da, C.F., Universidade Federal de Goiás, setembro de 2019. Bioprospecção de bactérias à partir da comunidade endofítica radicular cultivável de Aloe vera (L.) Burm. F. (Xanthorrhoeaceae). Orientador: Edson Luiz Souchie. Co-orientador: Marcos Antônio Soares.

A Aloe vera (L.) Burm. f., conhecida popularmente como babosa, está entre as plantas medicinais mais importante economicamente. Suas folhas possuem alta atividade biológica, entre seus importantes componentes destaca-se a aloína, capaz de fornecer matéria-prima para usos industriais como: medicinais, cosméticos, alimentos entre outros. Plantas em associação com micro-organismos benéficos, podem maximizar a produtividade e a qualidade dos produtos obtidos. Os micro-organismos endofíticos podem exercer diversas funções positivas às plantas, úteis na agricultura e na indústria, como substitutos de produtos químicos, com alto potencial biotecnológico. Com este trabalho, objetivou-se estudar, avaliar e selecionar bactérias endofíticas associadas às raízes de Aloe vera (L.) Burm. f. com potencial biotecnológico para a promoção do crescimento vegetal e aumento da produção de aloína. Foi realizado o isolamento, identificação molecular e seleção de micro-organismos endofíticos, realizada com base nos traços funcionais: produção de ácido indol-3-acético, solubilização de fosfatos de Bayóvar, cálcio, ferro e alumínio, e sideróforos, atividade antagônica a fitopatógenos e produção de enzimas extracelulares. Posteriormente, foram selecionadas quatro estirpes com potencial multifuncional dos três ambientes: campo, horta e viveiro. Os experimentos foram conduzidos em vasos, com seis tratamentos, onde foram inoculados nas mudas de A. vera os quatros isolados de forma individual, 389C Pantoea cypripedii, 35V Enterobacter ludwigii, 135V Enterobacter tabaci, 149H Paraburkholderia sp., uma mistura com todas as estirpes e o controle. As características de crescimento da planta e conteúdo de aloína foram avaliados após seis meses. As bactérias 149H Paraburkholderia sp. e 35V E. ludwigii, demonstraram papel relevante para incrementar a biomassa das plantas e conteúdo de aloína. Os mesmos podem ser utilizados como inoculantes, a fim de estabelecer um sistema de produção sustentável.

Palavras-chaves: Babosa, Micro-organismos, Traços funcionais, Promoção de crescimento vegetal, Biotecnologia, Aloína

ABSTRAT

SILVA, Cintia Faria da, C.F., Universidade Federal de Goiás, September 2019. Bioprospecting of bacteria from the cultivable root endophytic community of Aloe vera (L.) Burm f. (Xanthorrhoeaceae). Advisor: Edson Luiz Souchie. Co- advisor: Marcos Antônio Soares.

Aloe vera (L.) Burm. f., commonly known as aloe, is among the most economically important medicinal plants, having significant biocultural value. Its leaves have gel and latex, wich have expressive activity, complex chemical composition, components such as aloin and are able to provide raw material for uses such as: medicine, cosmetics, food industry among others. Plants are naturally associated with microorganisms, which interact in a mutualistic manner and can maximize the productivity and quality of the products obtained. Endophytic microorganisms have a variety of plant-beneficial functions and are useful in agriculture and industry as substitutes for chemicals with high biotechnological potential. This work aimed to study, evaluate and select an endophytic bacterial community associated with Aloe vera (L.) Burm f. roots wich has biotechnological potential to promote plant growth and increase aloin production. Isolation, molecular identification and selection of endophytic microorganisms from the plant itself were performed. This selection was made based on the functional traits: indolacetic acid production, Bayóvar phosphate solubilization, calcium, iron and aluminum, and siderophores, phytopathogen antagonistic activity and extracellular enzyme production. Subsequently, four strains with multifunctional potential were selected. The experiments were carried out in pots with six treatments, where A. vera seedlings were inoculated with four individual isolates, a mixture with all strains and the control. Plant growth parameters and aloin content were evaluated after six months. Bacteria 149H Paraburkholderia sp. and 35V E. ludwigii, demonstrated a relevant role in increasing plant biomass and aloin content. They can be used as inoculants in order to establish a profitable and sustainable production system.

Keywords: Aloe, Microorganisms, Functional traits, Plant growth promotion, Biotechnology, Aloin

1. INTRODUÇÃO GERAL

O Brasil possui uma grande biodiversidade da flora, além de um considerável conhecimento tradicional sobre o manejo e uso de plantas medicinais, o qual é passado de geração para geração, em que estas são utilizadas como remédios caseiros e matéria- prima para a fabricação de fitoterápicos e outros medicamentos (Leão et al., 2007). A Aloe vera (L.) Burm. f., conhecida popularmente como babosa, é uma planta de origem africana, pertencente à família Xanthorrhoeaceae, à qual pertencem mais de 500 espécies (Amoo et al., 2014), com ampla distribuição em regiões tropicais e subtropicais em todo o mundo (Patel e Patel, 2013). É uma das plantas medicinais biologicamente mais ativas, por isso, é considerada valiosa (Van Wyk, 2011; Patel e Patel, 2013; Zapata et al., 2013). Essa espécie fornece matéria-prima para diferentes usos industriais, tais como: medicinais, cosméticos, bebidas tônicas e inúmeros outros (Silva et al., 2013; Zapata et al., 2013). A parte da planta de maior importância industrial é a folha, que contêm um gel, cuja composição química é bastante complexa, composta principalmente de polissacarídeos, açúcares solúveis, proteínas, enzimas, aminoácidos, vitaminas e antraquinonas (Zapata et al., 2013), que são os constituintes mais importantes (Uikey et al., 2013). As antraquinonas (aloína, aloe-emodina) podem ter efeito contra o câncer, diabetes e inflamação, além de ações nos sistemas antioxidante, cardiovascular, metabólico e enzimático (Patel e Patel, 2013), o que levou a um aumento industrial e comercial na produção de A. vera em todo o mundo (Uikey et al., 2013). No ano de 2013, o Ministério da Saúde brasileiro, incluiu o uso da A. vera, como substância ativa em medicamento fitoterápico na lista da Assistência Farmacêutica do SUS (Sistema Único de Saúde), por meio do Programa Nacional de Plantas Medicinais e Fitoterápicos do Componente Básico da Assistência Farmacêutica. O uso nas formas de gel e creme é indicado para o tratamento tópico de queimaduras de 1º e 2º graus e Psoríase vulgaris, aprovado pela ANVISA (BRASIL, 2013), reconhecendo suas propriedades medicinais e importância. As plantas são naturalmente associadas a micro-organismos, que interagem de forma mutualística. Esta interação planta x micro-organismo pode, potencialmente, oferecer novas estratégias para melhorar a produtividade da planta, sem impacto negativo ao ambiente (Schenk et al., 2012).

Os micro-organismos endofíticos são aqueles que colonizam o tecido vegetal internamente, às vezes em número elevado, sem prejudicar ou causar doença ao hospedeiro (Reinhold-Hurek e Hurek, 2011). Estes endofíticos exercem diversas funções estratégicas nas plantas, tais como aumento na resistência aos estresses bióticos e abióticos, alteração de propriedades fisiológicas e produção de fitormônios, sendo potencialmente úteis na agricultura e na indústria, sobretudo na farmacêutica e de defensivos agrícolas. Portanto, os micro-organismos são potenciais substitutos de produtos químicos e possuem alto potencial biotecnológico (Santos e Varavallo, 2011). A seleção de cepas que possuem potencial multifuncional é estratégica, haja vista a existência de isolados com traços funcionais benéficos à planta (Vassilev et al., 2012). Isso é explicado pela liberação de metabólitos que estimulam diretamente o crescimento da planta, tais como: fitormônios, como auxinas, citocininas e giberelinas; inibição da produção de etileno; fixação de N2; solubilização de fosfatos inorgânicos e mineralização de fosfato orgânico e/ ou de outros nutrientes; além do antagonismo a micro-organismos fitopatogênicos; síntese de antibióticos; enzimas e competição com micro-organismos patogênicos (Esitken et al., 2010). Plantas de A. vera são de interesse econômico crescente, em função de suas propriedades medicinais e nutricionais (Araujo et al., 2002; Das et al., 2010), devido os potenciais fitoquímicos e farmacológicos (Amoo et al., 2014). A propagação convencional não é capaz de satisfazer tal demanda no mercado, que requer material vegetal saudável e homogêneo (Cristiano et al., 2016). Estratégias como o uso de micro- organismos selecionados podem acelerar tal processo, desse modo, proporcionar um melhor crescimento das plantas, com maior produção de biomassa vegetal e aumento no teor de aloína (Gupta et al. 2012). Com isso, há necessidade de bioprospectar e desenvolver novos processos e produtos neste campo (Van Wyk, 2011). Esse trabalho, visa investigar o potencial de micro-organismos endofíticos de raízes de A. vera, com caracteristicas para interagir e produzir efeitos benéficos sobre suas plantas, explorando sua capacidade para promoção do crescimento do vegetal, como aumento de biomassa e compostos bioativos, considerando uma planta medicinal de grande interesse comercial.

2. REVISÃO DE LITERATURA

2.1. Aloe vera O nome Aloe vera, cujo gênero deriva da palavra árabe "Alloeh", significa "substância amarga brilhante" e "vera", do latim "verdadeiro" (Surjushe et al., 2008). Uma planta cosmopolita, pertencente à família Xanthorrhoeaceae (Amoo et al., 2014), amplamente distribuída em regiões tropicais e subtropicais do mundo, que cresce principalmente em regiões secas da África, Ásia, Europa e América (Surjushe et al., 2008; Patel e Patel, 2013). A A. vera é originada da África do Sul (Radha e Laxmipriya, 2015), possui relatos de que suas folhas foram negociadas no comércio desde o século IV A.C, o que resultou no movimento da espécie ao longo de rotas comerciais da Península Árabe para o Mediterrâneo, subcontinente indiano, no Caribe e nas Américas, onde se tornou naturalizada (Grace, 2011).

Durante séculos, é utilizada por suas propriedades curativas e terapêuticas, considerada uma "panacéia universal", "a planta da imortalidade", aquela que tem a capacidade de curar qualquer tipo de doença (Radha e Laxmipriya, 2015; Ray et al., 2015). Há relatos na história milenar que era o segredo da beleza de Cleópatra, ou mesmo que era algum tipo de dom miraculoso dos deuses, capaz de curar praticamente qualquer doença, muito utilizada na medicina popular para a pele e outros distúrbios (Grindlay e Reynolds, 1986). No Brasil, sua distribuição geográfica ocorre em várias regiões do país, citada em levantamentos etnobotânicos realizados em diversos estados, como Rondônia (Lima et al., 2011), Piauí (Da Silva et al., 2017), Pará (Martins et al., 2005; Leão et al., 2007), Maranhão (Coutinho et al., 2002; Santos e Vilanova, 2017), Rio Grande do Norte (Mosca e Loiola, 2009; Freitas et al., 2012), São Paulo (Souza e Dória, 2016), Minas Gerais (Costa e Mayworm, 2011; Messias et al., 2015), Goiás (Zucchi et al., 2013; Santos et al., 2015), Mato Grosso (Almeida et al., 2014; De Souza Ferreira et al., 2015), Mato Grosso do Sul (Pereira et al., 2009), Paraná (Paula e Cruz-Silva, 2010), Rio Grande do Sul (Colet et al., 2015) entre outros. É uma planta xerófita, suculenta, herbácea e perene, disposta em roseta com raízes superficiais, suas folhas são lanceoladas, cerosas e possuem espinhos em sua margem, como forma de defesa mecânica e produz um exsudato de sabor amargo abaixo da superfície das folhas, que atua como defesa química contra herbívoros

(Cousins e Witkowski, 2012), suas flores são amarelas, agrupadas em um único tronco ereto, que pode chegar a um metro (Hazrati et al., 2017). Adaptada a áreas de baixa disponibilidade de água, seu tecido parenquimático possui um elevado teor de água, variando de 99-99,5% e seu material sólido de 0,5- 1,0%, o qual contém mais de 75 compostos potencialmente ativos, incluindo gordura, ácidos orgânicos, compostos fenólicos, enzimas, minerais, vitaminas (Radha e Laxmipriya, 2015), polissacarídeos (D-glicose e D-manose), taninos, esteróides, hormônios vegetais e aminoácidos (Patel e Patel, 2013). Através de usos etnomedicinais, bem como os estudos farmacológicos e fitoquímicos, tem-se observado a eficiência de A. vera em atividades como: anti- inflamatória, antimicrobiana, antiplasmódica/ antimalárica, antioxidante (Amoo et al., 2014), anti-tumorais, gastroprotetora e imunomoduladora (Lucini et al., 2013), além de benefícios para o sistema cardiovascular e na cicatrização de feridas (Patel e Patel, 2013). Acredita-se que essas atividades biológicas são atribuídas a uma ação sinérgica de todos os seus compostos, em vez de uma única substância química, já que os efeitos benéficos ainda não foram bem correlacionados com componentes individuais (Hamman, 2008; Lucini et al., 2013). O comércio derivado de seus produtos naturais é baseado principalmente em dois materiais obtidos a partir das folhas: o exsudato, usado em laxantes e mesófilo (gel), em produtos de uso tópico, doenças de pele ou ingeridos, a fim de aliviar dores digestivas e proporcionar o bem-estar geral (Grace, 2011), além de produtos cosméticos como cremes, limpadores faciais, loções, sabões, shampoos, bebidas com aloe, leite, sorvete e suplementos alimentares (Sánchez-Machado et al., 2017a). Os produtos à base do tecido foliar de A. vera movimentam um mercado global anual estimado em US $13 bilhões, segundo o International Aloe Science Council, o que vem despertando interesse de investidores (Schulz, 2012), está entre as culturas medicinais de maior importância econômica em todo o mundo (Grace et al., 2015).

2.2. Aloína Uma das principais substâncias bioativas de A. vera são as antraquinonas aloína e aloe-emodina (Amoo et al., 2014). A aloína é uma antraquinona-C-glicosídeo de ocorrencia natural (Das et al., 2015; Darini et al., 2015). Um composto de cor amarela, formado pela mistura de dois diastereoisômeros: aloína A e B (Patel e Patel, 2013), com propriedades laxante, usados em preparações farmacêuticas que podem causar alergia

6 em pessoas suscetíveis. Por isso, a aloína deve ser ausente em produtos alimentares a base de A. vera (Domínguez-Fernández et al., 2012). O conteúdo de aloína pode variar de concentração de acordo com as condições de crescimento das plantas (Sánchez-Machado et al., 2017b), as estações quentes no ano, proporcionam um aumento em seu teor, pela maior inicedência de luz, com efeito direto na biossíntese de metabólitos secundário (Zapata et al., 2013; Hazrati et al., 2017). No látex, encontra-se uma quantidade até 100 vezes maior de aloína, que no gel, podendo ainda variar suas concetrações entre amostras frescas, com maior conteúdo que amostras secas (Sánchez-Machado et al., 2017b). Na literatura, há poucos relatos do uso de micro-organismos como eleicitores em plantas de A. vera para uma maior produção de aloína, como Cardarelli et al. (2013) com o uso dos fungos micorrízicos arbusculares Glomus intraradices e G. mosseae, e Gupta et al. (2012) com a inoculação de bactérias rizosféricas, ambos com efeito positivo sobre a concentração de aloína.

2.3. Cultivo e propagação Plantas do gênero Aloe possuem características ecofisiológicas como resistência à seca, algum grau de suculência, bem como, o metabolismo do ácido crassuláceas - CAM (Cristiano et al., 2016; Oda e Erena, 2017). As plantas CAM são eficientes no uso da água na fotossíntese, particularidade associada à suculência da folha e do caule (Holtum et al., 2016). Economicamente, pode ser uma opção atraente aos agricultores de regiões com baixa precipitação (Sánchez-Machado et al., 2017a). A maior produção de biomassa foliar e o alto rendimento está associado a eficiência de uso da água (Silva et al., 2010), entretanto o estresse hídrico severo pode diminuir ou limitar o rendimento das folhas e o crescimento das plantas, contudo pode proporcionar um aumento de compostos bioquímicos e fitoquímicos (Hazrati et al., 2017). O alto nível de radiação pode aumentar a concentração de aloína, especialmente no verão (Zapata et al., 2013). Para a obtenção de uma alta produtividade por maior tempo, a expectativa é que cada planta deve emitir anuamente a mesma quantidade de folhas novas em relação as colhidas, geralmente em torno de 4 a 6 (Silva et al., 2010). A colheita das folhas pode iniciar após 7 a 8 meses do plantio, com o maior produtividade e rendimento satisfatório alcançados durante 4 a 5 anos, porém a duração de vida é de cerca de 12 anos (Sánchez- Machado et al., 2017a).

Tradicionalmente, em sistemas de produção convencionais, as plantas de A. vera são propagadas via brotos laterais emitidos da planta mãe, que varia de três a quatro no período vegetativo, assim, utilizado como materiais de plantio, porém, não é capaz de satisfazer a crescente demanda do mercado de aloe (Singh e Sood, 2009; Cristiano et al., 2016). A propagação de plantas de A. vera via sementes é possível somente pela polinização cruzada, o que envolve grande heterogeneidade da população de sementes (Cristiano et al., 2016), embora haja a produção de grande número de flores bissexuais saudáveis, muitas são inférteis (Das et al., 2015). O uso da técnica de micropropagação pode oferecer um grande potencial para a melhoria da planta, pela garantia qualidade, genética estável e padronizada, que podem ser regeneradas em grande número, independente de variáveis sazonais e ambientais, disponível durante todo o ano (Sahoo e Rout 2014; Das et al., 2015). A utilização de biorreatores de imersão contínua associado ao ozônio, pode eliminar o alto risco de contaminação no substrato líquido, sem a utilização de tecnologia sofisticada, com a rápida multiplicação e produção em grande quantidade de biomassa para extração de metabólitos secundários medicinais (Mariateresa et al., 2014). Segundo Cristiano et al. (2016), o custo de uma plantação micropropagada para os agricultores de países em desenvolvimento é muito alto, que pode variar entre 1,0 e 1,50 dolares por muda, o que torna a produção onerosa.

2.4. Micro-organismos endofíticos Registros fósseis datam que a mais de 400 milhões de anos as plantas são colonizadas por espécies microbianas, o que confirma que essas não vivem sob isolamento (Card et al., 2015). Essa interação entre organismos ocorre com a maioria das espécies de plantas e grupos de micro-organismos como os endofíticos, rizosféricos e os micorrízicos, os quais têm despertando grande interesse na comunidade científica devido seus benefícios (Reinhold-Hurek e Hurek, 2011; Card et al., 2015; Ludwig- Müller, 2015). Neste trabalho, foram enfocadas as bactérias endofíticas. Tais micro-organismos são capazes de colonizar os tecidos internos de plantas (Schulz e Boyle, 2005) sem causar qualquer sintoma aparente de doença (Qin et al., 2015), vivem em simbiose com a planta, onde a relação pode variar de saprofítico facultativo, parasita a mutualista (Schulz e Boyle, 2005).

As interações mutualistas entre plantas hospedeiras e micro-organismos associados, surge como resultado da seleção positiva exercida sobre essas associações, pois as plantas podem selecionar os micro-organismos mais competentes (Hardoim et al., 2008). Alguns aspectos podem determinar a estrutura da comunidade endofitica e sua associação com a planta, influenciada por fatores abióticos como local, temperatura e precipitação (Brum et al., 2012). A interação hospedeiro x micro-organismo pode variar sob certas condições, onde o endófito pode tornar-se parasita e vice-versa, sendo necessário um equilíbrio afinado entre as exigências do invasor (endofítico) e a resposta da planta, caso ocorra um desequilíbrio, uma doença pode se manifestar (Kogel et al., 2006). De acordo com Hardoim et al. (2008), a estratégia de vida das comunidades endofíticas podem ser classificadas como "obrigatória" ou "facultativa", em que os micro-organismos obrigatórios são estritamente dependentes da planta hospedeira e sua transmissão ocorre de forma vertical ou através de insetos vetores. Já os endófitos facultativos, considerados a maioria, caracterizam-se como bifásicos, isto é, alternam entre o interior da planta e o ambiente externo, especialmente o solo. Este comportamento sugere que a diversidade endofítica é um subconjunto da rizosfera, uma população associada às raízes (Santoyo et al., 2016). A colonização da planta hospedeira pode ocorrer através de fendas formadas em junções das raízes laterais, pela abrasão provocada pelo crescimento das raízes ao penetrar no solo, por feridas causadas por micro-organismos ou nematóides fitopatogênicos, ou ainda através de estômatos e hidatódios do tecido foliar (Hardoim et al., 2008; Santos e Varavalho, 2011) (Figura 1).

Figura 1. Tipos de endófitos e seu processo de colonização radicular (Hardoim et al., 2008). Conforme a Figura 1, as células em vermelho, são consideradas endofíticos passageiros, em sua maioria restritos ao tecido do córtex da raiz. Células azuis, são os endófitos oportunistas, limitam-se a determinados tecidos da planta e possuem características particulares para a colonização de raiz, com resposta quimiotática, o que lhes permite colonizar o rizoplano e depois invadir os tecidos internos da planta. Os endofíticos competentes, células em amarelo, são capazes de invadir tecidos específicos de plantas, tais como o tecido vascular, espalhando-se por toda a planta, sendo capaz de manipular seu metabolismo, o qual mantém um equilíbrio com a planta hospedeira, mesmo se presentes em alta densidade (Hardoim et al., 2008). Vários estudos através do isolamento e, ou identificação de endofíticos por técnicas convencionais (Brum et al., 2012; Bezerra et al., 2013; Qin et al., 2015) ou tecnologia sofisticada, como a metagenômica (Maropola et al., 2015; Akinsanya et al., 2015), têm evidenciado que as plantas são colonizadas por dezenas de micro- organismos, capazes de abrigar uma grande diversidade de espécies (Bezerra et al., 2013). Foram relatadas bactérias dos gêneros Azospirillum, Burkholderia, Klebsiella, Pseudomonas, Serratia (Santoyo et al., 2016), actinobactérias como Streptomyces, Gordonia, Nocardiopsis, Amycolatopsis, Nonomuraea, Micrococcus (Qin et al., 2015) e fungos Diaporthe, Phomopsis, Fusarium, Alternaria, Cladosporium, Acremonium, Trichoderma, Chrysonilia e Aspergillus (Bezerra et al., 2013; Martins et al., 2016).

Nas relações entre plantas e endofíticos é comum o fornecimento de nutrientes para os micro-organismos (Schulz e Boyle, 2005), enquanto as plantas são beneficiadas com a promoção do crescimento e a melhoria da resistência a estresses (Hardoim et al., 2008; Bezzera et al., 2013; Maropola et al., 2015), além da maior produção de compostos bioativos (Nisa et al., 2015). Os micro-organismos endofíticos utilizam mecanismos semelhantes aos dos rizosféricos para a promoção do crescimento da planta (Ali et al., 2014), o que proporciona benefícios diretos e indiretos. Esses podem ser, a produção de fitormônios, como o ácido indol-3-acético (AIA) (Dawwam et al., 2013), citocininas (Reinhold- Hurek e Hurek, 2011; Kudoyarova et al., 2014) e giberelinas (Piccoli et al., 2011; Khan et al., 2012). Além de atividades metabólicas variadas, como pela disponibilização de nutrientes através da solubilização de fosfatos (Dawwam et al., 2013), fixação de nitrogênio e sequestro de ferro pela produção e atividade de sideróforos (De Melo Pereira et al., 2012; Qin et al., 2015), ainda a atividade antagônica a fitopatógenos (Ma et al., 2013), além da produção de enzimas de interesse industrial (Corrêa et al., 2014) e produção de antibióticos (Christina et al., 2013). Durante a última década, os endófitos e seus produtos naturais bioativos ganharam crescente atenção por parte da comunidade científica devido ao seu potencial biotecnológico (Card et al., 2015; Nisa et al., 2015; Alvin et al., 2014). Alguns fungos endófitos são considerados importantes fontes de metabólitos bioativos (Bezerra et al., 2013) por sua capacidade em produzir os mesmos compostos de plantas, destacando-se como uma nova fonte para a produção de medicamentos (Nisa et al., 2015) e promoção de crescimento de plantas de interesse comercial (Qin et al., 2015; Joe et al., 2016; Verma et al., 2017).

2.5. Traços funcionais Os micro-organismos endofíticos estão dentro dos tecidos vegetais em contato com as células, o que lhes permite exercer de forma direta e com facilidade efeitos benéficos às plantas (Santoyo et al., 2016), como disponibilização de nutrientes, síntese de enzimas, de fitormônios, de sideróforos e antibiose contra fitopatógenos (Abbamondi et al., 2016). Os metabólitos produzidos por endofíticos têm sido amplamente estudados devido sua capacidade para melhorar o fornecimento de nutrientes e promover o crescimento vegetal; aumentar seu rendimento; reduzir os sintomas de doenças causadas

11 por agentes fitopatogênicos; reduzir a herbivoria de insetos e mamíferos; remover contaminantes do solo; melhorar o desempenho da planta sob condições ambientais de estresse, o que favorece o seu uso agrícola (Rashid et al., 2012; Card et al., 2015, Abbamondi et al., 2016). Recentemente, vem sendo estudado o potencial dos endofíticos em sintetizar produtos naturais bioativos, compostos miméticos aos metabólitos secundários associado às plantas, como fontes confiáveis a serem utilizadas direta ou indiretamente como agentes terapêuticos contra numerosas doenças (Kusari et al., 2014; Nisa et al., 2015). Alguns estudos com plantas medicinais de interesse comercial têm sido realizados com micro-organismos endofíticos, a fim de promover o crescimento e/ ou estimular maior produção de compostos bioativos de interesse (Nisa et al., 2015), como em Asclepias sinaica (Fouda et al., 2015), Phyllanthus amarus (Joe et al., 2016), Gynura procumbens (Bhore et al., 2010), Hyptis marrubioides (Vitorino et al., 2012), Jatropha curcas (Qin et al., 2015), com respostas promissoras. O crescimento e desenvolvimento vegetal é controlado por moléculas sinalizadoras, os chamados fitormônios, que também podem desempenhar respostas de defesa da planta (Ludwig-Müller, 2015). Esses fitormônios são auxinas, citocininas e giberelinas e outras biomoléculas, como o óxido nítrico ou metabólitos / enzimas, que interferem na síntese de etileno da planta (ACC-desaminase, jasmonatos e poliaminas), identificados em processos biológicos essenciais às plantas, como a divisão celular, alongamento, diferenciação, iniciação da raiz, floração, maturação e senescência (Kochar et al., 2011).

2.5.1. Síntese de auxinas Um dos mais importantes fitormônios, a auxina (ácido 3-indol acético ou AIA), que interfere diretamente no desenvolvimento do vegetal, são produzidos por plantas e por uma vasta gama de micro-organismos que usam a produção de AIA para interagir com plantas, como parte estratégica para a colonização, que inclui a fitoestimulação e uso deste fitormônio como molécula sinalizadora (Spaepen et al., 2007; Spaepen e Vanderleyden, 2011). A auxina tem impacto direto na fisiologia da planta, pois está envolvida na regulação da divisão celular, expansão de células e diferenciação celular, o que contribui para o desenvolvimento de órgãos como raízes laterais, brotos, folhas, flores e

12 frutos, além de estar relacionada na modelação vascular e tropismo. Pode atuar diretamente como uma molécula de defesa, com atividade antimicrobiana, a qual pode ser alterada por patógenos, para induzir sintomas de doença específicos durante o desenvolvimento da doença (Ludwig-Müller, 2015). Micro-organismos benéficos, interferem através da auxina no metabolismo da planta hospedeira para induzir o crescimento da planta em seu próprio benefício, com isso, afeta a resistência a vários grupos de agentes patogênicos, pois possui mecanismos moleculares semelhantes às vias de sinalização dos compostos associados com a defesa, como o ácido salicílico e ácido jasmônico (Ludwig-Müller, 2015; Fu et al., 2015). Os micro-organismos associados às plantas possuem diferentes rotas para biossíntese de AIA, sendo melhor estudada em bactérias (Spaepen et al., 2007). São descritas cinco vias dependentes do triptofano, pela expressão de genes como iaaM e iaaH, sendo estas: a via ácido indol-3-piruvato (IPyA); a via do indol-3-acetamida (IAM); a via indol-3-acetonitrilo (IAN); a via da triptamina (TAM); a via oxidase da cadeia lateral do triptofano (TSO) e uma via independente, em sua maioria similares às descritas em plantas, embora possa diferir por alguns intermediários (Spaepen et al., 2007; Kochar et al., 2011; Lin e Xu, 2013; Spaepen e Vanderleyden, 2011). A produção de AIA por micro-organismos endofíticos foi confirmada em diferentes gêneros bacterianos: Paecilomyces (Khan et al., 2012), Pseudomonas, Rhizobium, Agrobacterium (Abbamondi et al., 2016), Bacillus, Burkholderia (Dalal e Kulkarni, 2015), Herbaspirillum, Citrobacter, Pantoea, Acinetobacter (Joe et al., 2016), e fúngicos, tais como Trichoderma (Resende et al., 2013). Além da auxina, outros fitormônios como citocininas, giberelinas e etileno podem ter origem e regulação a partir de micro-organimos endofíticos e, igualmente, favorecer o crescimento, a nutrição e a produtividade vegetal (Bhore et al., 2010; Hamayun et al., 2010; Khan et al., 2012; Santoyo et al., 2016).

2.5.2. Solubilização de fosfatos Um traço funcional importante e bem desempenhado por micro-organismos é a solubilização de fosfatos. O fósforo (P) é um macronutriente essencial para as plantas, o segundo de maior importância, considerado limitante para o seu crescimento (Satyaprakash et al., 2017). Desempenha um papel importante em processos metabólicos, através do fornecimento de energia necessário para o metabolismo das plantas (Krishnakumar et al., 2014), fundamental na divisão celular, desenvolvimento,

13 quebra do açúcar, fotossíntese, transporte de nutrientes na planta, transferência de características genéticas entre gerações e regulação de vias metabólicas (Srinivasan et al., 2012; Behera et al., 2014). Embora o P seja um nutriente abundante no solo nas formas orgânicas e inorgânicas, possui baixa disponibilidade devido sua rápida fixação. Aproximadamente 95-99% de P no solo está presente sob a forma de fosfatos insolúveis (Krishnakumar et al., 2014). Também pode ser encontrado de forma imobilizada na matéria orgânica, que representa um importante reservatório, com 20-80% e apenas 0,1% do total de P existente no solo está disponível na forma solúvel e disponível às plantas (Bashan et al., 2013). No Brasil, as frações de P nos solos são encontradas em maior abundância em forma de fosfato de ferro (Fe-P), seguida de fosfato de alumínio (Al-P) e fosfato de cálcio (Ca-P), todos como baixa solubilidade e decresce na seguinte ordem: Ca-P > Al- P > Fe-P (Barroso e Nahas, 2008). Em solos ácidos, a fixação ocorre sob a forma de fosfato de ferro e fosfato de alumínio, já em solos neutro-alcalino sob a forma de fosfato de cálcio (Srinivasan et al., 2012). Diversos micro-organismos possuem artifícios capazes de converter o P insolúvel em formas solúveis, através de mecanismos como a produção de ácidos orgânicos ou enzimas fosfatases (ácida e/ ou alcalina) (Krishnakumar et al., 2014), com - -2 a liberação de formas de H2PO 4 ou HPO4 , que podem ser absorvidas pelas plantas (Behera et al., 2014). Entre os micro-organismos solubilizadores de fosfato encontram- se bactérias dos gêneros Acinetobacter, Bacillus, Pseudomonas e Rhizobium e fungos, tais como Alternaria, Aspergillus, Fusarium, Helminthosparium, Penicillium entre outros (Behera et al., 2014; Joe et al., 2016).

2.5.3. Produção de sideróforos O ferro é um micronutriente essencial para o metabolismo das plantas, abundante na natureza, porém de forma oxidada (Fe3+), com baixa solubilidade, comumente encontrado sob forma de hidróxido insolúvel (Benite et al., 2002). Entretanto, os micro-organismos podem driblar tal limitação nutricional, através da produção e utilização de quelantes, os chamados sideróforos, do grego: "transportadores de ferro" (Neilands, 1995), uma das principais fontes de ferro para as plantas (Sujatha e Ammani, 2013).

Os sideróforos são moléculas de proteínas de baixo peso molecular (Fazary et al., 2016) com alta afinidade e especificidade para Fe III (Boukhalfa e Crumbliss, 2002) de uma classe de agentes quelantes biológicos produzidos por micro-organismos capazes de promover a absorção de nutrientes metálicos inacessíveis (Andrews et al., 2016). Possuem cerca de 500 compostos distintos, cuja função é absorver o ferro mediado pelas células microbianas (Boukhalfa e Crumbliss, 2002), sendo divididos em três classes funcionais principais: hidroxamatos, catecolatos/ fenolatos e hidroxicarboxilatos (Fazary et al., 2016). A biossíntese de sideróforos ocorre sob condições de baixo teor de ferro (Butler e Theisen, 2010), já em condições de elevadas concentrações, a produção desses compostos é reprimida (Machuca e Milagres, 2003). Os sideróforos contribuem para o antagonismo a fitopatógenos (Sujatha e Ammani, 2013), pois quelam o ferro livre presente no solo, tornando-o indisponível para micro-organismos deletérios (Fazary et al., 2016).

2.5.4. Controle biológico - Antibiose O constante uso de pesticidas químicos acarreta graves dilemas, como riscos de segurança à saúde humana e animal, redução da biodiversidade, poluição ambiental e resistência de patógenos (Zhang et al., 2016). Como alternativa para minimizar tais problemas, pode-se utilizar o controle biológico, uma prática chave na agricultura sustentável para o controle de doenças por fitopatógenos (Lin et al., 2014), porém é um ramo que tem sido pouco explorado (Evangelista-Martinez, 2014). Bettiol et al. (2012), em documento publicado pela Embrapa Meio Ambiente, retrataram parte do mercado mundial dos agentes de biocontrole de doenças de plantas disponíveis, com informações de apenas 135 produtos à base de micro-organismos, o que demostra a necessidade de mais estudos. O controle biológico através do uso de micro-organismos nativos antagonistas é uma alternativa promissora para proteger as culturas contra doenças, dada a diversidade microbiana dos ecossistemas do solo (Evangelista-Martinez, 2014), além disso, muitos desses são eficazes na promoção do crescimento da planta (Lin et al., 2014). A inibição de fungos patogênicos e a indução de resistência de plantas hospedeiras pela produção de vários compostos antibióticos, também pode ocorrer pela produção de enzimas extracelulares degradadoras da parede celular, tais como quitinase,

15 celulase, protease e β-(1-3) glucanase, que tem papel importante na inibição dos agentes fitopatogênicos (Singh et al., 2011; Saravanakumar et al., 2016). O emprego de micro-organismos não patogênicos a seus hospedeiros tem se mostrado eficaz no controle de fitopatógenos (Rocha et al., 2009), como o fungo Trichoderma harzianum (Zhang et al., 2016), bactérias dos gêneros Bacillus (El- Bendary et al., 2016; Zouari et al., 2016), Paenibacillus, Pseudomonas (Lin et al., 2014) e leveduras tais como Pichia ohmeri e Candida guilliermondii (Coelho et al., 2011), isoladas a partir de plantas, inclusive medicinais (Xiang et al., 2016). Esses micro- organismos desempenham um papel importante, pois podem expressar boas atividades antagônicas em condições in vitro e ex vitro, associados com a eficiência na promoção do crescimento da planta (Lin et al., 2014). A supressão de um agente patogênico ou praga por antagonistas em um sistema de cultivo, pode resultar na diminuição da incidência e severidade de doenças (Pliego et al., 2011), que pode ser conseguido pela utilização de agentes microbianos específicos, pela introdução de micro-organismos selecionados no sistema (Rocha et al., 2009). O gênero Bacillus oferece vantagens sobre outras bactérias, pois é atóxico, inofensivo para os seres humanos e animais, além de não ser fitopatogênico (Yang et al., 2009). Produz alguns metabólitos de interesse, principalmente lipopéptidos cíclicos (iturinas, fengicinas e surfactinas) que agem de forma sinérgica, caracterizados por uma baixa toxicidade, elevada biodegradabilidade e características protetoras ao ambiente, com expressiva atividade inibidora sobre os vírus, bactérias, fungos, oomicetos e mosquitos (Zouari et al., 2016). Esses organismos exibem uma forte atividade antifúngica e um amplo espectro de antibióticos, além da capacidade de formar endósporos, o que facilita o armazenamento a longo prazo e, consequentemente, a comercialização (Yang et al., 2009). Outro gênero importante é o das Pseudomonas, produtoras de antibióticos, presentes em diversos ambientes como rizosfera, filosfera, de fácil isolamento e manipulação genética, o que favorece a experimentação (Raaijmakers et al., 2002). Entre os fungos, o gênero Trichoderma é bastante conhecido entre os agentes de biocontrole contra fitopatógenos, o qual é atualmente comercializado com mais de 100 compostos antimicrobianos identificados (Bae et al., 2016). Segundo Rocha et al. (2009) e Lin et al. (2014), os micro-organismos antagonistas são submetidos a testes de atividade antimicrobiana, em condições de laboratório ou casa de vegetação para aplicação de populações que expressam

16 atividades antagônicas satisfatórias, a fim de investigar o seu potencial para aplicação agrícola.

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3. JUSTIFICATIVA

O gênero Aloe é mundialmente reconhecido pelo seu valor medicinal (Amoo et al., 2014), com mais de 200 substâncias biologicamente ativas (Radha e Laxmipriya, 2015), entretanto vem sendo cada vez mais utilizado em uma diversidade de produtos, desde cosméticos, medicamentos, alimentos, produtos de limpeza e até mesmo na indústria de colchões. Mundialmente, o mercado de extratos de A. vera vêm crescendo o longo dos anos segundo o site Statista - The Statistics Portal, um dos bancos de dados estatísticos mais bem-sucedidos do mundo, as vendas em 2016 totalizaram cerca de 1,6 bilhões de dólares e prevê para 2021 um aumento para 2,3 bilhões de dólares (Statista, 2018), o que caracteriza um mercado promissor. Os agricultores brasileiros pouco tem explorado o cultivo de plantas de A. vera, porém a indústria necessita cada vez mais de matéria prima para suprir a demanda do mercado. Propriedades rurais de pequeno e médio porte, são uma boa opção para investir em culturas não tradicionais, como A. vera (Cristiano et al., 2016). O pequeno produtor, tem seu lugar na cadeia produtiva, com a obtenção de lucro considerável, através da agricultura familiar, especialmente quando há o respaldado de uma empresa parceira, como uma agroindústria por exemplo, pela industrialização e comercialização da produção (Bach e Lopes, 2007). Usualmetente, os produtores de A. vera não utilizam fertilizantes, a fim de reduzir o custo da produção em campo (Cristiano et al., 2016), mas o uso micro- organismos promotores de crescimento com potencial para fitoestimulação, biofertilização e, ou biocontrole (Abbamondi et al., 2016), podem contribuir efetivamente na produtividade, fornecendo para o mercado matéria-prima de qualidade, com maior volume, sendo assim um benefício para os agricultores. Esporádicos trabalhos são realizados com micro-organismos para incrementar a biomassa e teor de aloína das plantas de A. vera.

A introdução de bactérias promotoras de crescimento vegetal em plantas alvo, pode colaborar para o aumento da produtividade, e a indução de compostos bioativos importantes (Timmusk et al., 2017) e sobretudo a melhoria da qualidade do solo (Ambrosini et al., 2016). As associações simbióticas entre tais bactérias e as plantas, permite aumentar a biodisponibilidade de nutrientes essenciais como fósforo, nitrogênio, entre outros, indispensáveis para o crescimento das plantas, além de melhorar a absorção de água, modular a produção fitohormônios e diminuir de efeitos provocados por estresses bióticos e abióticos (Basu et al., 2017).

4. REFERÊNCIAS BIBLIOGRÁFICAS

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OBJETIVO GERAL

Estudar, avaliar e selecionar bactérias endofíticas associadas às raízes de Aloe vera (L.) Burm. f. com potencial biotecnológico para a promoção do crescimento vegetal e aumento da produção de aloína.

OBJETIVOS ESPECÍFICOS

- Isolar micro-organismos endofíticos de raízes de Aloe vera; - Selecionar micro-organismos produtores de ácido indolacético; - Quantificar a capacidade de solubilização de fosfatos; - Testar a atividade antagonista in vitro com fungos fitopatogênicos e produção de sideróforos; - Avaliar a produção de enzimas extracelulares e tolerância a salinidade. - Eleger bactérias com potencial multifuncional para inoculação em plantas de A. vera; - Avaliar o efeito da inoculação sobre os parâmetros biométricos no aumento da biomassa da planta, através da inoculação de bactérias benéficas; - Quantificar a produção de aloína em plantas submetidas a inoculação de bactérias.

CAPÍTULO I

Artigo aceito pela revista South African Journal of Botany

Screening of plant growth-promoting endophytic bacteria from the roots of the medicinal plant Aloe vera

Abstract This work aimed to test the hypothesis that the root bacteria of A. vera present multifunctionality with potential in biotechnology for plant growth. A total of 129 endophytic bacteria from three environments, a field, a garden, and nursery were isolated and evaluated for indole acetic acid-producing ability. Thirty-two of the total bacteria isolates were evaluated for siderophore production, phosphate solubilization and antibiosis to phytopathogenic fungi. The phylogenetic analysis revealed the presence of four groups: Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. The functional trait for indole acetic acid (IAA) synthesis was observed in 32 of the isolates, with emphasis on the 135V Enterobacter tabaci strain, which revealed the highest production (225.2 μg mL-1). The results found for Bayóvar phosphate solubilization were not expressive, with the highest values observed for the bacterium 149H Paraburkholderia sp. (45.7 mg L-1). The 3V isolate Brevibacillus agri presented 45.6% antagonism against S. sclerotiorum. The greatest inhibition of the phytopathogens Fusarium sp. and Rhizoctonia sp. was observed with 149H Paraburkholderia sp. and 348V E. tabaci, respectively. This was the first study to evaluate the potential of endophytic bacteria of A. vera for plant growth promotion. Our results indicate that the isolates 135V and 348V E. tabaci and 149H Paraburkholderia sp. have potential for to be field-tested as growth promoter inoculants.

Keywords Endophytes, Auxin, Phosphate solubilization, Antibiosis, Functional traits, Plant growth promotion.

1. Introduction

Plants establish intrinsic relationships with a range of microorganisms, such as endophytic, rhizospheric, and mycorrhizal organisms, which have aroused the interest of the scientific community owing to their proven benefits (Reinhold-Hurek and Hurek, 2011; Card et al., 2015; Ludwig-Müller, 2015; Cordero et al., 2017; Liotti et al., 2018). These microorganisms are naturally associated with the host plant and can occur endogenously. This is true for endophytic microorganisms, which colonize internal plant tissues for at least a part of their life-cycle (Hardoim et al., 2015) without causing functional damage or disease symptoms (Schulz and Boyle, 2005; Reinhold-Hurek and Hurek, 2011; Zawoznik and Groppa, 2019). In addition, they may be dependent on the host plant, through vertical transmission or vector insects (Hardoim et al., 2008). Endophytic bacterial communities are dynamic and capable of spreading systemically throughout plant tissues (Bacon and White Jr, 2015). Such colonization has several benefits, since this community can communicate and interact efficiently, regardless of the environmental conditions to which the plant is subjected (Santoyo et al., 2016). This increases plant growth (Khamwan et al., 2018) and maximizes protection against plant pathogens (Zhao et al., 2018), which can enhance resistance to stress (Sharma et al., 2015; Kumar and Verma, 2018) and may stimulate the synthesis or production of bioactive compounds of interest (Alvin et al., 2014; Venugopalan and Srivastava, 2015; McMullin et al., 2018). Several studies have confirmed that bacterial strains associated with roots can promote plant health and growth through mechanisms such as phytostimulation, biofertilization, and/or biocontrol (Verma et al., 2011; Gaiero et al., 2013; Abbamondi et al., 2016). Several genera, such as Pseudomonas (Ali et al., 2014; Liffourrena and Lucchesi, 2018), Pantoea, Bacillus (Andreolli et al., 2016), Serratia, Enterobacter (Otieno et al., 2013), Azospirillum (Hungria et al., 2018), and Paraburkholderia (Bernabeu et al., 2018) are beneficial for plant growth. The association between endophytic plants and microorganisms results in direct physiological effects on plant growth and development, such as: nitrogen fixation (Joe et al., 2016) and phosphate solubilization (Andreolli et al., 2016; Pande et al., 2017; Lobo et al., 2019), as well as the production of ammonia (Yaish et al., 2015), siderophores (Abbamondi et al., 2016), phytohormones (De Melo Pereira et al., 2012; Lin and Xu, 2013), and hydrolytic enzymes (Joe et al., 2016). These benefits meet the

38 needs of current plant cultivation through sustainable methods that reduce the use of chemical fertilizers and pesticides, replacing conventional mechanisms to preserve soil biological diversity. Therefore, a strategic alternative is the selection and use of microorganisms with biotechnological application, which influences plant growth and health, as well as soil quality and nutrient cycling. Knowledge about plant-soil relationships and the deleterious effects that excessive or inadequate application of chemical fertilizers have on the environment have changed farmers' views regarding the use of biological products, such as microbial inoculants; their use has increased globally owing to their proven agronomic effects (Malusá and Vassilev, 2014). The production of P fertilizers is expensive because they are based on the chemical processing of insoluble rock phosphate, which makes access to farmers under-capitalized and typically family-based, onerous. Thus, biotechnology- based on phosphate-soluble bioinoculants have been proposed as alternatives to increase P availability to crops and reduce fertilizer use (Busato et al., 2017). Bacteria, fungi, and actinomycetes isolated from medicinal plants such as Asclepias sinaica (Fouda et al., 2015), Echinacea purpurea, Lonicera japonica (Gupta et al., 2012), Rauwolfia serpentina, Gymnema sylvestre, Stevia crenata, Bacopa monnieri, Andrographis paniculata, Withania somnifera (Singh et al., 2016), Teucrium polium (Hassan, 2017), Terminalia bohera, and Manihot esculenta (Ansary et al., 2018), have been shown to be efficient in agriculture, and have been applied to different crops owing to the multiple functions they perform. Medicinal plants act in a specific manner when selecting endophytes, since the choice may be based on the secondary metabolites produced by the plant and the composition of the root exudates; therefore, these microbial communities diversify according to their nutritional needs, the type of soil, and the environment in which they are found (Maggini et al., 2018). Studies have shown that bioactive compounds synthesized by host medicinal plants are also produced by the endophytic microbiota (Abdou et al., 2010; Venieraki et al., 2017; Danagoudar et al., 2018; Gupta et al., 2018; Tan et al., 2018; Kaaniche et al., 2019), and there is evidence that metabolic synthesis pathways have evolved independently in plants and microorganisms (Jensen et al., 2011; Hamayun et al., 2017), and for horizontal gene transfer (Richards et al., 2009). Metabolites other than those produced by the medicinal plant can also be produced by the endophytic microbiota, contributing to the heterogeneity of the phytochemical profile and plant biofunctions (Aly et al., 2008; Nisa et al., 2015; Nakaew and

Sungthong, 2018). Therefore, medicinal plants may represent readily available sources of microorganisms with biotechnological potential (Gupta et al., 2012; Passari et al., 2015). Aloe vera L. Burm. f. is a plant known worldwide for traditional medicine and is of significant commercial importance (Zapata et al., 2013; Radha and Laxmipriya, 2015). It is widely used for the production of cosmetics, tonics, and in the food industry (Zapata et al., 2013), and has great medicinal potential, with approximately 75 active ingredients described (Bjørklund et al., 2018). Considering that this potential may be due in part to the endophytic community of A. vera, we tested the hypothesis that the root endophytic bacteria of this plant were multifunctional, which could contribute biotechnologically to agriculture. Recently, Akinsanya et al. (2015a) isolated endophytic bacteria from A. vera, including Pseudomonas hibiscicola, Macrococcus caseolyticus, Enterobacter ludwigii, and Bacillus anthracis, which can produce bioactive compounds of medical importance with antimicrobial activity against bacterial pathogens. In another study, Akinsanya et al. (2015b) evaluated the diversity of the endophytic bacterial community of this plant by metagenomics, and demonstrated a greater colonization in the root system; however no studies have examined the functional traits expressed by this community. Therefore, to address this gap, we aimed to isolate cultivable bacterial isolates associated with the roots of A. vera obtained from heterogeneous environments, and to test the biotechnological potential of the strains in terms of plant growth.

2. Material and methods

2.1. Study area and sample collection The root systems of three A. vera individuals were sampled in different environments in the city of Rio Verde, GO, central-western Brazil, in the following locations and geographical coordinates: (1) nursery (latitude 17°47.224'S, longitude 050°57.966'W, and altitude of 786 m), in interaction with diverse vegetation, between arboreal and herbaceous species; (2) garden (latitude 17°48.137'S, longitude 050°55.824'W, and altitude of 701 m) in contact with herbaceous plants, especially medicinal plants, receiving periodic cultural treatments; and (3) field (latitude 18°13.407'S, longitude 51°01.174'W, and altitude of 553 m), anthropic region, of little vegetation (Fig.1). Sampling was performed with the objective of recovering a wide

40 diversity of cultivable bacteria, increasing the chances of isolating bacteria with functional traits for biotechnological application. To avoid that climatic factors could affect the dynamics of the microbial community, interfering with the results, all A. vera plants were sampled on the same day (02/05/2015), between 08 and 10 AM. This was a rainy day, and the temperature between the collection areas varied between 19 and 20 ºC, while the relative air humidity remained at 80%. The individuals selected had no apparent disease symptoms. The collected roots were stored in properly identified plastic bags and transported to the Agricultural Microbiology Laboratory of IF Goiano, Rio Verde Campus for analysis.

Fig. 1. Environments from which Aloe vera roots were collected. A) Field: Farm; B) Garden of the Planta e Vida Cooperative; C) Nursery of the University of Rio Verde, GO; D) Extensive root-system of A. vera.

2.2. Endophytic bacteria isolation Endophytic bacteria were isolated using root fragments. These were washed in running water to remove excess adhered soil and fragments were then shaken in water and neutral detergent (1%) at 70 rpm for 10 min to reduce the density of epiphytic microorganisms. The surface of the fragments was disinfected by successive washes in ethanol (70%), 2.5% sodium hypochlorite (active chlorine), and ethanol (70%) for 1

41 min, 5 min, and 30 s, respectively. At the end of the process, four washes were performed using autoclaved distilled water. In addition, an aliquot of 100 μL was taken from the final wash for inoculation in a nutrient broth (3 g meat extract, 5 g peptone) at 28°C for 24 h to test the efficiency of the disinfestation process. The disinfested fragments were cut to approximately 1 cm in length and placed on Petri dishes containing potato dextrose agar (Acumedia®; PDA). The growth of endophytic bacteria was monitored until day 10. The frequency of colonization was evaluated considering the percentage of fragments with at least one endophytic bacteria, in relation to the total fragments analyzed, according to the formula below.

Frequency of colonization = (No. fragments/ Total fragments) x 100

2.3. Spore extraction of arbuscular mycorrhizal fungi (AMF) Three samples of rhizospheric soil were collected from the same sampling sites at 0–20 cm depth. Spores were extracted by the wet-sieving method (Gerdemann and Nicolson, 1963), whereby 50 g of dry soil was passed through 0.42 and 0.053 mm mesh sieves and centrifuged at 1,811x g in water and at 804x g in a solution of 50% sucrose, for 3 and 2 mins, respectively. The spore density was determined by counting under z Zeiss Discovery V8 (40x) stereomicroscope on a plate with canalettes.

2.4. Identification and phylogenetic relationships of isolates Total genomic DNA was extracted from the purified bacteria as described by Cheng and Jiang (2006). Bacterial species were identified based on partial sequencing of the 16S rRNA, using the universal primers 27F (5'-AGA GTT TGA TCM TGG CTCAG-3') and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3') (Weisburg et al., 1991). PCR products were purified using the PureLink Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen®) as instructed by the manufacturer. Sequencing was performed by the Sanger method. Fragments with 16S gene sequences of at least 900 bp were analyzed by the software BLASTn, and each strain was identified through the National Center for Biotechnology Information Blast (www.ncbi.nlm.nih.gov/BLAST) (Altschul et al., 1990). The phylogenetic relationships of the isolates were inferred using 661 SNPs found in the sequences obtained from the 16S regions aligned with type sequences

42 extracted from the database of the Ribosomal Database Project and the National Center for Biotechnology Information Blast using the software CLUSTAL OMEGA (Sievers et al., 2011). The reference sequences were chosen based on the degree of similarity to the sequences obtained from the isolates, given by the databases. The evolutionary model of the TrN+G sequences was selected using Bayesian Information Criterion (BIC), implemented in software JMODELTEST 2 (Darriba et al., 2012). The phylogenetic tree was inferred through the software MR BAYES v.3.2.6. (Ronquist et al., 2012), using methods based on Bayesian inference. Four independent runs were performed, with 10 × 106 generations assigned to each chain, with probability distribution a posteriori for every 500 generations. Before calculating the consensus tree, and to ensure the convergence of the chains, the first 2,500 trees sampled were discarded. Subsequently, the recovered phylogeny was tested by the bootstrap method, with 5,000 replications, through the MEGA 7 program (Kumar et al., 2016). The tree obtained was visualized and edited with the program FigTree v 1.4.2 (Rambaut, 2014). A sequence from Methylobacterium rhodinum was used as an out-group.

2.5. Selection of endophytic bacteria and auxin production Indoleacetic acid (IAA) production was used to investigate the multifunctional potential of bacteria; only bacteria with this potential were tested for other traits. The use of this criterion reflects the fact that root endophytic bacteria are associated with the biosynthesis of this phytohormone, and can directly affect its homeostasis, while rhizosphere microorganisms tend to be more associated with nutrient supply (Marasco et al., 2012). IAA production was quantified using the colorimetric method developed by Gordon and Weber (1951). The bacterial isolates were cultured under constant stirring at 90 rpm in nutrient broth medium (3 g meat extract, 5 g peptone) at 28°C for 72 h. The optical density (OD600) of all bacterial samples was adjusted to 0.3 by dilution with saline solution (0.85%). IAA production was quantified in triplicate. Bacterial cultures were inoculated in nutrient broth medium supplemented with L-tryptophan (1%) and maintained for 72 h at 28°C in the dark at 90 rpm. As a control, the broth culture medium was used with tryptophan without bacterial inoculum. After incubation, the samples were centrifuged (12,000 rpm for 5 min at 10°C), then 1 mL of the supernatant from each isolate was

43 transferred to a test tube, and 1 mL of Salkowski reagent (1,875 g FeCl3.6H2O, 100 mL

H2O, and 150 mL H2SO4) was added. The tubes were kept in the dark for 15 min at 28°C for further quantification of IAA in a spectrophotometer (530 nm). IAA concentrations were obtained using the equation of the calibration curve obtained with commercial IAA (De Melo Pereira et al., 2012). The 32 strains with the most satisfactory results for IAA production were then tested for other functional traits.

2.6. Phosphate solubilization

Bacterial cultures were standardized at an OD600 of 0.3 and inoculated in triplicate, with 1 mL in each penicillin glass containing 9 mL of GY culture medium (10 g glucose, 2 g yeast extract), supplemented separately with four phosphate sources: 5g L-1 of reactive natural phosphate from Bayóvar, Peru (12.8% P), 5g L-1 from tricalcium 2 -1 -1 phosphate (Ca3(PO4) ), 2g L from iron phosphate (FePO4), and 1g L of aluminum phosphate (AlPO4). These remained under constant agitation at 90 rpm for 72 h at 28°C. As a control, the GY medium was used with each phosphate source. Subsequently, the pH was measured. The amount of inorganic P was determined by the colorimetric method described by Murphy and Riley (1962). Bacterial phosphate solubilization was estimated using the standard curve equation.

2.7. Siderophore production Siderophore production was evaluated using the universal methodology adapted by Schwyn and Neilands (1987). The bacterial isolates were cultured in tryptophanin soybean culture medium, diluted 1/10, and incubated in an oven at 28°C for 72 h. The cell suspension was centrifuged at 12,000 rpm for 10 min, 1 mL of the supernatant was transferred into test tubes, and then 1 mL of chromoazide S (CAS) indicator solution was added. Conversion of the blue color of the CAS solution in the supernatant to orange-yellow over the 15-min period indicated that the isolate was capable of producing siderophores.

2.8. Antibiosis to phytopathogenic fungi The antagonism of bacteria against the phytopathogens Sclerotinia sclerotiorum (white mold), Fusarium sp., and Rhizoctonia sp. was tested using the double culture method (Mew and Rosales, 1986). Initially, four endophytic bacteria were inoculated at

44 the edge of each plate containing PDA culture medium, 3 cm from the center of the plate, where a 5 mm diameter pathogen mycelium disc was deposited. For the control treatment, a plate containing the phytopathogen in the center was used. Evaluations were made relative to the control plates, where mycelial growth was observed up to 3 cm away from the center, by visual analysis. Isolates presenting some degree of phytopathogen inhibition were individually tested in triplicate. In a Petri dish containing PDA medium, a 5 mm diameter culture dish containing endophytic bacterium and each pathogenic isolate was inoculated at equal distance. The control treatment consisted of a plate containing only the phytopathogen. The plates were incubated at 25 ºC until the mycelium of the pathogen developed, without the presence of the endophytic bacteria, on the whole culture medium. Subsequently, the zone of inhibition provoked by the different endophytic bacteria was evaluated. The diameter of each fungus was measured with a pachymeter and the zone of fungal growth inhibition was confirmed by the production of suppressive compounds by the bacterium. The percentage suppression by each treatment was calculated using the relative index (RI), as follows: RI(%)=(RC−RX)/ RC×100, where: RC = radius of the pathogen colony in the control treatment RX = radius of the pathogen colony paired with the endophytic isolate

2.9. Enzymatic evaluation The enzymatic activity (amylase, cellulase, pectinase, and protease) of the strains was evaluated. A pre-inoculum was performed in which bacteria were grown in nutrient broth culture medium for 48 h, at 90 rpm at room temperature. For the tests, 5 μL of each isolate was inoculated by the micro-plating technique in the culture medium used for each test, and four strains were evaluated per Petri dish.

2.9.1. Amylase production To determine amylolytic activity, the bacteria were inoculated by micro-plating in nutrient agar (NA) medium supplemented with 0.2% soluble starch (Hankin and Anagnostakis, 1975) and incubated for 72 h at 28°C. The culture medium was then covered with a Lugol solution for 15 min. Following exposure to metallic iodine, a

45 transparent halo was observed around colonies that produced amylase (Strauss et al., 2001).

2.9.2. Cellulase production To determine cellulase production, the culture medium was used as described by Cattelan (1999), whereby the bacteria were inoculated in trypticasein soy agar medium (TSA) containing 10 g/L cellulose and incubated for 72 h at 28°C. A Congo red solution (0.3%) was added to each plate for 15 min and incubated at room temperature. The excess was then removed and NaCl (1M) solution was added for discoloration and incubation for 15 min. Cellulase-producing bacteria presented a light-colored halo with orange borders, indicative of hydrolysis (Teather and Wood, 1982).

2.9.3. Pectinase production To confirm the production of pectinases, the method proposed by Hankin et al. (1971) was modified. Briefly, PDA medium plus 10 g of pectin/L was used. Bacteria were grown for 48 h at 28°C and the plate was covered for 15 min with hexadecyltrimethylammonium bromide solution (1%). This reagent precipitates intact pectin in the medium, and light zones around the colony indicate the degradation of pectin via the action of pectinase.

2.9.4. Protease production YPD medium (5 g peptone, 3 g yeast extract, 10 g glucose, and 20 g agar containing 2% casein) was used, and solubilized in hot water followed by autoclaving for 10 min at 110°C. The plates were incubated for 72 h at 28°C. Bacteria were cultivated in this medium; protease-positive bacteria formed a light halo around their colonies, indicating the presence of proteases (Strauss et al., 2001).

2.10. Tolerance to salinity Four bacteria, which presented positive characteristics for plant growth in previous tests, were selected. The tolerance of these bacteria was evaluated in nutrient agar medium containing different concentrations of NaCl (0, 1, 2, 4, and 6%) and incubated for 48 h at 28°C, as described by Cardoso et al. (2017).

2.11. Statistical analyses The tests were conducted in a completely randomized design, always in triplicate. Data were submitted to analysis of variance and the average AMF spore densities were compared by Tukey’s test (5%). Average values for phosphate solubilization, IAA synthesis, and antibiosis were compared by the Scott-Knott test (5%). All analyses were performed using the software SISVAR (Ferreira, 2011).

3. Results

3.1. Isolation and density of endophytic bacteria and AMF spores The colonization frequency of endophytic bacteria in the root fragments was 100% for the nursery and garden samples and 98.6% for the field sample. In total, 129 endophytic bacteria were obtained, including 36 from the field, 20 from the garden, and 73 from the nursery. Only 32 isolates were selected for subsequent studies based on auxin production as follows: one from the field, six from the garden, and 25 from the nursery (Fig. 2).

Fig. 2. Percentage of endophytic bacteria isolated from the roots of A. vera. Bacteria were selected based on the production of indoleacetic acid and arbuscular microrrheic fungi (AMF) spores on the rhizospheric soil of the samples field, garden, and nursery. ( ) number of bacteria and number of spores.

The density of soil spores differed significantly between the environments evaluated; the density was highest in the nursery samples at 1,375 (50 g-1) compared with the garden samples at 865 (50 g-1) and field samples at 93 (50 g-1).

3.2. Identification and phylogenetic analysis of isolates The identified bacteria represented 10 genera, distributed in four groups. Proteobacteria was the most abundant and was represented by the classes Gammaproteobacteria and Betaproteobacteria; Firmicutes, class Bacilli; Bacteroidetes, class Flavobacteriia; and Actinobacteria, class Actinobacteria. The 32 isolates were identified by the degree of genetic similarity in: Enterobacter sp. (4), Enterobacter asburiae (1), Enterobacter tabaci (8), E. ludwigii (4), Pantoea agglomerans (1), P. cypripedii (1), Lelliottia nimipressuralis (1), Paraburkholderia sp. (1), Bacillus megaterium (3), B. agri (1), Lysinibacillus xylanilyticus (1), L. macroides (2), Microbacterium aerolatum (1), and Chryseobacterium taiwanense (3) (Fig. 3).

Fig. 3. Similarity tree based on 16S gene sequences from endophytic bacteria isolated from the roots of A. vera in three different sites with relationships between the nearest strains. (T) Type strain. Letters following the isolate numbers indicate the sampling site (V = nursery, H = garden, and C = field). Black numbers below the nodes indicate the posterior probability, and the blue values above the nodes represent the bootstrap values based on 10,000 replicates.

3.3. IAA production and selection of endophytic bacteria The ability to synthesize IAA was evaluated quantitatively in 129 endophytic bacteria; 24.8% (32 bacteria) presented positive results, with concentrations varying between 0.3 and 225.2 μg mL-1. Nineteen bacterial isolates produced significant amounts of this phytohormone (greater than 80 μg mL-1) and only seven isolates presented less than 20 μg mL-1 of IAA. The 135V E. tabaci isolate produced the highest concentrations of IAA (Table 1).

Table 1 In vitro production of indoleacetic acid (IAA) by bacteria obtained from the roots of Aloe vera.

-1 Isolate IAA (μg mL ) σM ± 389C Pantoea cypripedii 42.4 d 1.5 62H Enterobacter sp. 113.7 b 10.3 63H Enterobacter sp. 116.3 b 6.7 65H Enterobacter sp. 119.4 b 8.8 149H Paraburkholderia sp. 132.6 b 1.9 180H Enterobacter sp. 97.9 c 4.5 383H Lysinibacillus macroides 5.7 d 1.6 2V Bacillus megaterium 6.4 d 1.9 3V Brevibacillus agri 0.3 d 0.2 4V Bacillus megaterium 5.3 d 2.6 6V Lysinibacillus xylanilyticus 7.9 d 1.2 14V Enterobacter tabaci 119.2 b 1.4 16V Lysinibacillus macroides 29.9 d 1.9 17V Enterobacter tabaci 83.0 c 8.6 18V Enterobacter tabaci 113.3 b 6.5 20V Enterobacter tabaci 88.6 c 10.8 23V Microbacterium aerolatum 11.6 d 6.7 32V Chryseobacterium taiwanense 24.3 d 1.2 35V Enterobacter ludwigii 122.8 b 3.7 38V Enterobacter asburiae 152.1 b 0.7 41V Bacillus megaterium 8.3 d 1.2 43V Enterobacter tabaci 111.6 b 12.0 49V Enterobacter ludwigii 83.2 c 11.1 54V Pantoea agglomerans 20.7 d 1.0 116V Chryseobacterium taiwanense 22.1 d 0.5 125V Chryseobacterium taiwanense 24.3 d 2.9 126V Enterobacter ludwigii 101.0 c 8.7 127V Enterobacter ludwigii 115.9 b 3.1 128V Enterobacter tabaci 131.0 b 6.8 135V Enterobacter tabaci 225.2 a 2.9 348V Enterobacter tabaci 103.4 c 1.6 349V Lelliottia nimipressuralis 104.0 c 1.8 Average values followed by the same letter do not

3.4. Phosphate solubilization Maximum Bayóvar phosphate solubilization was detected by the bacterium 149H Paraburkholderia sp. (45.7 mg L-1), which reduced the pH to 3.3. The most effective solubilization of Ca3(PO4)2 was observed with the isolate 389C Pantoea 49

-1 cypripedii (36.5 mg L ), with pH 3.9. For FePO4, only four bacteria demonstrated solubilization, although the levels observed were low, at ≤ 3.4. When using AlPO4 as a phosphate source, no solubilization was observed for any of the bacteria tested. Isolates 383H Lysinibacillus macroides, 6V and 16V L. xylanilyticus, and 116V and 125V C. taiwanense did not solubilize any of the phosphate sources tested. As expected, lower final pH values were related to higher solubilization levels (Table 2).

Table 2. pH and in vitro solubilization of Bayóvar natural phosphate (P), tricalcium phosphate (Ca3(PO4)2), and iron phosphate (FePO4) by A. vera root endophytic bacteria.

Bayóvar phosphate Ca3(PO4)2 ¤ FePO4 Isolate -1 -1 -1 pH P soluble (mg L ) σM ± pH P soluble (mg L ) σM ± pH P soluble (mg L ) σM ± 389C Pantoea cypripedii 6.3 c - - 3.9 a 36.5 a 0.18 3.4 a 2.8 a 0.02

62H Enterobacter sp. 5.2 b 7.0 c 0.09 6.7 e 4.9 g 0.03 7.0 f - -

63H Enterobacter sp. 5.3 b 5.7 c 0.11 6.8 e 2.1 h 0.02 7.2 f - -

65H Enterobacter sp. 4.4 a 10.3 b 0.09 6.8 e 2.0 h 0.01 6.9 f - -

149H Paraburkholderia sp. 3.3 a 45.7 a 0.12 6.1 d 29.6 b 0.29 7.0 f - -

180H Enterobacter sp. 5.0 b 12.9 b 0.03 6.8 e 2.1 h 0.01 8.0 f - -

383H Lysinibacillus macroides 8.1 d - - 8.0 f - - 7.0 d - -

2V Bacillus megaterium 4.1 c 2.6 d 0.04 5.5 c 8.7 f 0.03 5.6 d - -

3V Brevibacillus agri 7.5 d - - 7.1 e - - 4.2 b 2.1 a 0.03

4V Bacillus megaterium 4.5 a - - 4.8 b 23.8 c 0.06 4.8 c - -

6V Lysinibacillus xylanilyticus 7.5 d - - 8.0 f - - 7.8 f - -

14V Enterobacter tabaci 6.8 c - - 6.6 e 8.4 f 0.02 7.1 f - -

16V Lysinibacillus macroides 6.4 c - - 8.2 f - - 5.8 d - -

17V Enterobacter tabaci 6.9 c - - 6.7 e 9.1 f 0.06 7.1 f - -

18V Enterobacter tabaci 4.8 b 3.0 d 0.03 4.9 b 21.5 d 0.03 5.3 d 0.7 b 0.02

20V Enterobacter tabaci 6.7 c - - 6.7 e 5.9 g 0.03 6.7 e - -

23V Microbacterium aerolatum 6.0 c - - 5.3 c 18.2 d 0.07 5.7 d - -

32V Chryseobacterium taiwanense 6.9 c - - 6.0 d 6.1 g 0.02 5.6 d - -

35V Enterobacter ludwigii 5.3 b 12.3 b 0.09 6.6 e 12.6 e 0.05 7.1 f - -

38V Enterobacter asburiae 6.7 c - - 6.6 e 4.1 g 0.04 7.0 f - -

41V Bacillus megaterium 4.1 a 2.1 d 0.01 4.9 b 25.6 c 0.12 5.4 d - -

43V Enterobacter tabaci 6.7 c - - 6.5 e 6.7 g 0.05 7.2 f - -

49V Enterobacter ludwigii 4.3 a 16.9 b 0.09 6.0 d 3.3 h 0.02 6.0 d - -

54V Pantoea agglomerans 5.1 b 7.1 c 0.08 6,1 d 24.6 c 0.05 4.8 c 3.4 a 0.01

116V Chryseobacterium taiwanense 6.6 c - - 6.2 d - - 5.5 d - -

125V Chryseobacterium taiwanense 6.9 c - - 6.2 d - - 5.5 d - -

126V Enterobacter ludwigii 4.5 a 9.0 b 0.01 6.5 e 14.5 e 0.18 6.6 e - -

127V Enterobacter ludwigii 5.2 b 11.5 b 0.16 6.7 e 3.8 h 0.01 6.8 e - -

128V Enterobacter tabaci 6.6 c - - 6.7 e 2.4 h 0.03 7.0 f - -

135V Enterobacter tabaci 4.4 a 13.0 b 0.01 6.7 e 9.0 f 0.13 5.6 d - -

348V Enterobacter tabaci 4.4 a 14.0 b 0.05 6.9 e 2.4 h 0.07 5.7 d - -

349V Lelliottia nimipressuralis 4.3 a 5.0 c 0.04 6.6 e 5.5 g 0.18 6.3 e - -

Averages followed by the same letter do not differ from one another by the Scott-Knott test (5%). (-) indicates no solubilization; σM = standard error of the mean.

3.5. Antibiosis and siderophore production Initial screening with 32 bacteria revealed that 40.6% (13) of isolates exerted some degree of mycelial growth inhibition of S. sclerotiorum, 34.4% (11) on Fusarium sp., and 21.9% (7) against Rhizoctonia sp. (Fig. 4). Quantitative assessment of antagonistic activity against S. sclerotiorum revealed a variation of 11.4 to 45.6% in terms of relative inhibition. Among the isolates, 383H L. macroides, 3V Brevibacillus agri, and 14V and 17V Enterobacter tabaci presented inhibition rates between 35.6 and 45.6%. For the phytopathogens Fusarium sp. and Rhizoctonia sp., the relative inhibition was lower, reaching no more than 34%, with the highest levels presented by the bacteria 149H Paraburkholderia sp. and 348V Enterobacter tabaci (Table 3). Siderophore production was negative for all bacterial isolates evaluated.

Fig. 4. Double culture test between root endophytic bacteria of A. vera and phytopathogens Sclerotinia sclerotiorum, Fusarium sp., and Rhizoctonia sp. (A) S. sclerotiorum (control); (B) Antagonism between the phytopathogenic fungus S. sclerotiorum and the bacterium 14V Enterobacter tabaci. (C) Fusarium sp. (control); (D) Antagonism between Fusarium sp. and the bacterium 149H Paraburkholderia sp. (E) Rhizoctonia sp. (control); (F) Antagonism between Rhizoctonia sp. and the bacterium 348V Enterobacter tabaci. Microscopic images showed relevant morphological changes after 4 days of S. sclerotiorum phytopathogen cultivation with endophytic bacteria. Some hyphae became swollen, twisted, and dehydrated, and others were collapsed (Fig. 5).

Fig. 5. Morphological alterations of the mycelium of the phytopathogenic fungus S. sclerotiorum are indicated by red arrows following interaction with root endophytic bacteria of A. vera. (A) Control - hyphae of S. sclerotiorum; (B) interaction with the 3V B. agri bacterium – hyphae swollen; (C) 14V Enterobacter tabaci bacterium - hyphae twisted; (D) bacterium 17V E. tabaci - degenerative changes in the morphology of hyphae; (E) 383H L. macroides bacteria – hyphae collapsed or swollen.

Table 3. Inhibition of mycelial growth of phytopathogenic fungi Sclerotinia sclerotiorum, Fusarium sp., and Rhizoctonia sp. by endophytic bacteria in antagonism tests in vitro. Sclerotinia Fusarium sp. Rhizoctonia sp. sclerotiorum Isolate 1 2 1 2 1 2 RC σM (RC) RI RC σM (RC) RI RC σM RI

(cm) ± (%) (cm) ± (%) (cm) (RC) ± (%) 389C Pantoea cypripedii 8.0 c 0.07 11.4 8.0 b 0.07 11.5 - - - 62H Enterobacter sp. ------63H Enterobacter sp. - - - 8.4 b 0.08 6.3 7.1 b 0.04 21.5 65H Enterobacter sp. - - - 8.8 b 0.05 1.9 8.8 c 0.11 2.2 149H Paraburkholderia sp. 6.5 b 0.13 28.1 6.1 a 0.05 32.2 - - - 180H Enterobacter sp. 7.0 c 0.05 21.9 ------383H Lysinibacillus macroides 5.3 a 0.50 41.4 ------2V Bacillus megaterium ------3V Brevibacillus agri 4.9 a 0.06 45.6 ------4V Bacillus megaterium ------Lysinibacillus 6V ------xylanilyticus 14V Enterobacter tabaci 5.1 a 0.17 43.0 8.1 b 0.04 10.4 - - - 16V Lysinibacillus macroides ------17V Enterobacter tabaci 5.8 a 0.23 35.6 ------18V Enterobacter tabaci 7.3 c 0.11 19.2 ------20V Enterobacter tabaci ------Microbacterium 23V 7.8 c 0.23 13.7 ------aerolatum Chryseobacterium 32V ------taiwanense 35V Enterobacter ludwigii 6.2 b 0.12 31.4 ------38V Enterobacter asburiae - - - 8.7 b 0.00 3.3 7.7 b 0.37 14.1 41V Bacillus megaterium ------43V Enterobacter tabaci ------49V Enterobacter ludwigii - - - 8.1 b 0.23 10.0 7.1 b 0.36 21.5 54V Pantoea agglomerans 7.2 c 0.07 19.7 7.9 b 0.35 11.9 - - - Chryseobacterium 116V ------7.1 b 0.04 21.5 taiwanense Chryseobacterium 125V ------taiwanense 126V Enterobacter ludwigii ------127V Enterobacter ludwigii - - - 8.9 b 0.06 1.1 - - - 128V Enterobacter tabaci - - - 8.4 b 0.06 6.7 8.9 c 0.04 0.7 135V Enterobacter tabaci ------348V Enterobacter tabaci 6.6 b 0.17 27.0 6.8 b 0.00 24.4 6.0 a 0.28 33.7 349V Lelliottia nimipressuralis 6.8 b 0.03 24.8 ------Control 9.0 d 0.00 0.0 9.0 b 0.00 0.0 9.0 c 0.00 0.0 Averages followed by the same letter do not differ by the Scott-Knott test (5%); 1Radius of phytopathogen colony. 2Relative inhibition. (-): indicates no inhibitory activity; σM = standard error of the mean.

3.6. Enzymatic evaluation and salinity tolerance There was evidence of amylolytic activity in 34.4% (11) of the bacteria by bacterial isolates 2V, 4V, and 41V (B. megaterium), 3V (B. agri), 18V and 135V (E. tabaci), 23V (M. aerolatum), 32V, 116V, and 125V (C. taiwanense), and 349V (L. nimipressuralis). No cellulase, protease, or pectinase activity was observed for any the bacteria tested. With respect to salinity, among the selected bacteria, 35V E. ludwigii, 135V E. tabaci, and 389C P. cypripedii presented good growth under all NaCl concentrations, while 149H Paraburkholderia sp. did not tolerate the highest concentrations of NaCl (4 and 6%).

3.7. Multifunctional potential of isolates None of the 32 isolates tested have all the 13 functional characteristics evaluated. Only three isolates were able to produce IAA, and six of the functional characteristics evaluated were observed in three bacteria (Fig. 6A). The predominant features were IAA production and calcium phosphate solubilization (Fig. 6B).

Fig. 6. Multifunctional potential of endophytic bacteria associated with the roots of A. vera from different sites. A) Number of bacterial isolates expressing potential functional traits for biotechnology. B) Number of isolates presenting each functional trait evaluated (IAA biosynthesis, amylase production, Bayóvar phosphate solubilization, calcium and iron, and antibiosis to phytopathogens S. sclerotiorum, Fusarium sp., and Rhizoctonia sp.).

Isolates 149H Paraburkholderia sp., and 135V and 348V E. tabaci were notable for their expression of the functional traits evaluated (Fig. 7). The 135V isolate

55 produced the highest levels of IAA detected, and showed potential for Bayóvar phosphate solubilization, amylase production, and salinity tolerance, while the 149H isolate produced the second-highest level of IAA, and the highest values for Bayóvar phosphate solubilization, and relative inhibition of Fusarium sp. The 348V isolate showed good potential for IAA synthesis, Bayóvar phosphate solubilization, and phytopathogen inhibition, especially Rhizoctonia sp.

Fig. 7. Endophytic bacteria 149H Paraburkolderia sp., and 135V and 348V Enterobacter tabaci isolates from the roots of A. vera and functional traits expressed by these isolates. Arrows indicate that the isolate expresses the indicated trait.

4. Discussion Despite the high interest in plants used for traditional medicine, little is known about the symbiotic associations of these plants with endophytic microorganisms (Alvin et al., 2014; Andreolli et al., 2016; Pereira et al., 2016). The present study was the first to evaluate the multifunctional potential of bacterial strains isolated from the roots of A. vera.

4.1. Phylogenetic characterization of isolates High bacterial colonization and a high number of AMF spores were observed in the nursery. In this environment, A. vera plants were associated with a wide diversity of plants. According to Silva et al. (2014), AMF density can be explained by the wide

56 variety of vegetable species; thus, systems with high vegetation diversity present high values for this index. The results of this study demonstrated that the characteristics of the environment affect the functional diversity of bacterial communities. For example, 36 endophytic bacteria were isolated from the field, which has scarce vegetation, but only one of those species (Pantoea cypripedii) was able to produce IAA and present other functional traits. From the garden, where the specimen was closely associated with a variety of medicinal plants, 20 bacteria were isolated, of which six strains of Enterobacter sp., Paraburkholderia sp., and Lysinibacillus macroide presented both the initial trait for IAA production, as well as other traces evaluated. From the nursery, which is rich in arboreous and herbaceous species, a higher number of isolates (73) were obtained, and 25 of those had functional traits, represented by the species P. agglomerans, Enterobacter sp., E. tabaci, E. ludwigii, E. asburiae, L. nimipressuralis, B. megaterium, B. agri, L. xylanilyticus, L. macroides, M. aerolatum, and C. taiwanense. We identified a high number of isolates from the groups Proteobacteria and Firmicutes, followed by Actinobacteria and Bacteroids. The predominance of these groups was also observed by Akinsanya et al. (2015b), who performed a metagenomic study to identify endophytic bacteria in tissues such as the leaves, stem, and roots of A. vera. In our samples, we identified bacteria that have previously been reported as growth promoters, with potential for biostimulation, biocontrol, and biofertilization (Bernabeu et al., 2018; Numan et al., 2018; Sun et al., 2018; Zhao et al., 2018). These bacteria belong to the genera Bacillus (Haque et al., 2016; Munjal et al., 2016), Paraburkholderia (Bolívar-Anillo et al., 2016; Bernabeu et al., 2018), Enterobacter (Dolkar et al., 2018; Zhao et al, 2018), Lysinibacillus (Kumar et al., 2017), Microbacterium (Sun et al., 2018) and Pantoea (Trifi et al., 2017). Some genera of the family Enterobacteriaceae are polyphyletic, including Enterobacter and Paraburkholderia, which are currently under revision, and thus hindered the allocation of new related species using only the 16S rRNA gene sequence. One example is the bacterium L. nimipressuralis, which was formerly classified as Enterobacter (Brady et al., 2013). The genus Paraburkholderia, containing growth- promoting species, was also recently renamed. This previously belonged to the genus Burkholderia, a group that contains opportunistic pathogens that are harmful to human and animal health (Dobritsa and Samadpour, 2016). The α- and β-Proteobacteria classes are related to nitrogen fixation (Estrada-De Los Santos et al., 2016; Liotti et al., 2018).

The presence of the genera Enterobacter, Pantoea, and Paraburkholderia in the roots of A. vera may play an important role in nitrogen absorption in this plant.

4.2. Multifunctional Isolates Potential In this study, the genus Enterobacter represented 54% of the isolates, with species demonstrating capacity for IAA production, Bayóvar phosphate, calcium and iron solubilization, and antibiotic activity against the phytopathogens S. sclerotiorum, Fusarium sp., and Rhizoctonia sp. Similar results were observed by Nutaratat et al. (2017) with the strain Enterobacter sp. DMKU-RP206, which showed great potential for IAA production, phosphate solubilization, ammonia and siderophores production, and antagonism against the pathogenic rice fungi Curvularia lunata, Rhizoctonia solani, and Fusarium moniliforme. Members of the Firmicutes phylum produce a range of antimicrobial metabolites, enzymes, and surfactants that promote growth and induce systemic resistance in plants (Bibi, 2017). Bacteria of the genus Bacillus have demonstrated resistance to metals and can be used in phytoremediation. In addition, they produce enzymes such as cellulases, amylases, and xylanases. Bacteria of the genus Microbacterium, belonging to the phylum Actinobacteria, have also shown potential for the synthesis of these enzymes, and for the solubilization of phosphates (Román-Ponce et al., 2017). Bacteriodetes, represented here by the bacterium Chryseobacterium taiwanense, have been reported to have the potential for biotechnological applications, including the production of enzymes and pigments (Tai et al., 2006; Wu et al., 2013; Zhao et al., 2015; Puentes-Téllez and Salles, 2018). The strains evaluated in this study were able to produce amylases and the phytohormone IAA. Auxin was produced by 32 of 129 bacteria tested, with emphasis on 135V E. tabaci, a species for which there is limited literature. This was recently described as a new member of the genus, and isolated from the stem of a tobacco plant (Duan et al., 2015). The levels of IAA synthesized varied between 0.3 and 225.2 μg mL-1, whereby the maximum values were significantly higher than those observed for endophytic bacteria obtained from other medicinal species such as Hyptis marrubioides (Vitorino et al., 2012), Phyllanthus amarus (Joe et al., 2016), and T. polium (Hassan, 2017). Variations in the levels of IAA can be explained by the location of genes responsible for biosynthesis, and the locations of these genes modulate the levels of this

58 phytohormone. When the genes are located on chromosomal DNA, they result in lower production; however, when they are located on the plasmid, they result in greater auxin production, as several copies of the gene are present (Spaepen and Vanderleyden, 2011). The relationship between phosphate solubilization and culture medium acidification was confirmed, since the microbial solubilization of P in organic and inorganic soils is generally associated with the release of low-weight molecular organic acids. Through hydroxyl and carboxyl groups, these chelate the cations bound to phosphate, thereby converting it to the soluble form (Behera et al., 2014). These organic acids may include acetate, lactate, malate, oxalate, succinate, citrate, and gluconate (Vassilev et al., 2012). Other mechanisms of P solubilization include H+ excretion and acid phosphatase biosynthesis (Satyaprakash et al., 2017). Gupta et al. (2012) evaluated rhizospheric bacteria of Aloe barbadensis using Pseudomonas synxantha, Burkholderia gladioli, Enterobacter hormaechei, and Serratia marcescens and observed tricalcium phosphate solubilization between 340 and 150 mg L-1. These levels are high, when compared with those obtained in the present study using A. vera endophytic bacteria. We observed solubilization levels ranging from 45.7 -1 -1 to 2.1 mg L for Bayóvar phosphate, 36.5 to 2.1 mg L for Ca3(PO4)2, and 3.4 to 0.7 for

FePO4. These levels indicate that solubilization is not the main functional trait of the analyzed strains. Another important functional trait evaluated in the present study was antibiosis to phytopathogens. The genus Brevibacillus showed potential for the inhibition of S. sclerotiorum. This phytopathogen is a cosmopolitan necrotrophic that attacks a range of hosts, including more than 400 plant species (Kabbage et al., 2015), such as Phaseolus vulgaris (Rocha et al., 2009), Glycine max (Zhang et al., 2016), and Brassica napus (Chen et al., 2014). In the present study, 3V B. agri induced 45.6% inhibition, which induced morphological changes in phytopathogenic hyphae (Fig. 5). Mohanty et al. (2017) isolated endophytic bacteria from Jatropha curcase and observed a predominance of the genus Brevibacillus, with positive characteristics for IAA production and phosphatases, which induced the growth of maize seedlings. In the present study, the B. agri species showed low potential for IAA synthesis and phosphate solubilization, but significantly suppressed the phytopathogen S. sclerotiorum. In a study by Yue et al. (2015), Brevibacillus inhibited the growth of S. sclerotiorum, and the formation of sclerotia, and Joo et al. (2015) confirmed the effect of rhizospheric

Brevibacillus on several species of Fusarium. Strains of this genus are considered potential candidates for biocontrol agents, owing to antibiotics production and biofilm formation (eg, Panda et al., 2014; Shaf et al., 2017; Arrigoni et al., 2018). Brevibacillus brevi strain FJAT-0809-GLX, as evaluated by Jianmei et al. (2015), inhibits the proliferation of pathogens through the production of the antimicrobial substance ethylparaben, in addition to chitinase, with the suppression of seven types of pathogens: Ralstonia solanacearum, Salmonella typhimurium, Escherichia coli, Fusarium oxysporum, Aspergillus niger, Fusarium solani, and Fusarium moniliforme. In this context, the class Bacilli is the most representative among the bacteria capable of inhibiting the growth of phytopathogenic fungi, consistent with many antagonist strains (Tumbarski et al., 2014; Andreolli et al., 2016; Francisco et al., 2016; Zouari et al., 2016; Douriet-Gámez et al., 2018). This is because the bacteria of this class synthesize antimicrobial substances, such as ethylparaben, and enzymes that attack the cell walls of phytopathogenic fungi, such as chitinases (Jianmei et al., 2015), and lipopeptides with antifungal properties (Torres et al., 2017; Toral et al., 2018; Villegas- Escobar et al., 2018). Thus, several studies confirmed the potential of Bacilli as biocontrol agents and encourage their use in agriculture (Jianmei et al., 2015). Conversely, the phytopathogen Fusarium sp. was inhibited to a greater extent by the bacterium 149H Paraburkholderia sp. with 32.2% RI. Huo et al. (2018) also observed the inhibitory activity of a Paraburkholderia rhizosphere isolate on root rot fungus. However, the phytopathogen Rhizoctonia sp. was mainly inhibited by the endophytic 348V E. tabaci, with 33.7% RI (Fig. 4). As it is a recently described species, additional data is not available in the literature confirming the ability of this bacterium to inhibit phytopathogens. In addition to IAA synthesis, most of the strains tested (91%) expressed other functional traits of biotechnological importance. Therefore, our fundings support the hypothesis that root endophytic bacteria from A. vera display multifunctionality, and some have biotechnological potential. Some of the functional traits can act in synergism, favoring the growth and development of the vegetable and improving the health and performance of the crop. Among the functional traits evaluated, the indices obtained by the multifunctional 135V and 348V isolates, which are both Enterobacter tabaci, were notable. Although this is a recently described species, Salkar et al. (2018) isolated a strain of this species with great biotechnological potential, suggesting its use in sustainable agriculture systems. Another notable isolate in relation to the tested

60 indices was 149H Paraburkholderia sp.; this genus contains environmental bacteria, including promising candidates for biotechnological applications (Bernabeu et al., 2018; Silva et al., 2018; Lobo et al., 2019). In vivo mechanisms for the use of this genus as inoculants have already been evaluated and positive effects on growth and productivity have been observed (e.g. Bernabeu et al., 2018; Rahman et al., 2018). Therefore, in this study, we have confirmed the in vitro biotechnological potential of several endophytic strains of A. vera, especially 135V, 348V, and 149H. This was the first study to evaluate the root endophytic bacterial community of A. vera, providing opportunities for the future use of endophytic bacteria of this species as inoculants for the promotion of vegetal growth.

5. Conclusions This study demonstrated the multifunctionality of endophytic root-endophytic bacteria of A. vera, and 24.8% of the isolates tested positive for IAA synthesis. 40.6% had some degree of inhibition of S. sclerotiorum, 34.4% of Fusarium sp. and 21.9% Rhizoctonia sp. Especially the isolates 135V and 348V Enterobacter tabaci and 149H Paraburkholderia sp., which expressed a set of functional traits with potential application for plant growth. Future in vivo assessments should aim to confirm the potential of these strains for use as inoculants, biofertilizers, or biological control agents of phytopathogens in order to replace chemicals and increase crop growth, health, and productivity.

Acknowledgements The authors thank the Instituto Federal Goiano - Campus Rio Verde for providing the infrastructure and materials to carry out this work.

Conflict of interests The authors declare no conflicts of interest.

Funding This work was supported by the Pro-Centro-Oeste Network and the Foundation for Research Support of the State of Goiás (FAPEG) and the Coordination for the Improvement of Higher Education Personnel (CAPES).

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CAPITULO II

Artigo de acordo com as normas da revsita South African Journal of Botany

Endophytic bacteria promote growth and increase the aloin content of Aloe vera

ABSTRACT Aloe vera (L.) Burm f. has enormous biocultural value, and is among the world’s economically most important medicinal plants. A number of sectors, in particular, the food and pharmaceutical industries, assemble a vast range of products derived from A. vera, with a global market worth US $13 billion. The application of endophytic microorganisms, selected for their multifunctional potential as inoculants, may contribute not only to an increase in the biomass of the plant, but also to its aloin content. This work aimed to investigate the application of endophytic bacteria with potential for both the promotion of growth in A. vera and the stimulation of the production of aloin. The experiment was based on the cultivation of A. vera plants in plastic pots. The seedlings were initially inoculated with four strains of endophytic bacteria (35V Enterobacter ludwigii, 135V Enterobacter tabaci, 149H Paraburkholderia sp., 389C Pantoea cypripedii) or a combination of the four strains. The results indicate that endophytic bacteria obtained from the roots of A. vera may play an important role in the growth and productivity of the plant, and that they can be used as inoculants to establish a sustainable agricultural system of high productivity. In particular, the bacterium 149H Paraburkholderia sp. was capable of increasing the growth of the aerial structure of A. vera and its gel content, as well as increasing the aloin content of the gel. The bacterium 35V Enterobacter ludwigii promoted an increase in the aloin content of both the gel and latex of the A. vera plants.

Key words: Medicinal plants; Inoculants; Micro-organisms; Productivity; Phytochemistry.

1. Introduction Aloe vera (L.) Burm f. is a species of the family Xanthorrhoeaceae (Amoo et al., 2014) and is not only among the world’s most economically important medicinal plants, but also has enormous biocultural value (Grace, 2011). According to the International Aloe Science Council, products derived from the leaves of A. vera support a global market of US $13 billion, which attracts the interest of investors worldwide (Schulz, 2012). Aloe vera is planted widely in warm, dry regions (Hazrati et al., 2017), where it grows primarily on well-drained soils (Cristiano et al., 2016). The plants of the genus Aloe have CAM (Crassulacean Acid Metabolism) photosynthesis, which minimizes water loss, as well as being capable of storing water in their leaves, which allows them to survive droughts (Cousins and Witkowski, 2012). Aloe leaves have a waxy surface, which reflects excess sunlight and minimizes evaporation through the stomata and external cells (Oda and Erena, 2017). The leaf has three layers – gel, latex, and cuticle. The gel is the most internal layer, and is formed by soft, mucilaginous tissue, which is transparent and lubricous, with large parenchymal cells, containing 99% water, and composed of glucomannans, amino acids, lipids, sterols, and vitamins (Surjushe et al., 2008). The latex is an intermediate layer, with a bitter yellow sap that contains anthraquinones and glycosides (Hamman, 2008). The cuticle is the thick outer layer, that forms a protective skin, in which carbohydrates and proteins are synthesized (Maan et al., 2018). The root system grows only a few centimeters below the surface of the soil, and absorbs water efficiently, even in areas with low precipitation (Cousins and Witkowski, 2012). The traditional ethnomedicinal use of A. vera, and the more recent pharmacological and phytochemical research, which has investigated the plant’s many associated active compounds, such as amino acids, sugars, enzymes, vitamins, minerals, saponins, anthraquinones, lignin, and salicylic acid (Misir et al., 2014), indicate that the plant has benefits in the treatment of a variety of diseases. The chemical composition of A. vera is highly diversified, and the plant is rich in secondary metabolites, which are mostly associated with the gel found inside the leaves, with more than 75 biological properties (Radha and Laxmipriya, 2015; Bjørklund et al., 2018). The most important compounds are aloin (barbaloin), emodin, isobarbaloin, aloe-emodin, aloesin, aloeresin, isoaloeresin, and acemannan (Hamman, 2008; Surjushe et al., 2008; Domínguez-Fernández et al., 2012; Baruah et al., 2016; Salah et al., 2017). However, the potential of A. vera is thought to be derived from the

82 synergic action of its compounds rather than a single chemical substance (Hamman, 2008; Lucini et al., 2013; Radha and Laxmipriya, 2015). Just as the soil may influence the physiology of a plant and affect the biochemical interactions between the roots and the soil microbiota, plants may select and recruit potential endophytic microorganisms through differences in the root architecture and chemical signals exuded by the root tissue. This may have a direct effect on the plant growth, through its influence on metabolic processes, an improvement in the supply of nutrients, the release of phytohormones, and protection against phytopathogens (Rashid et al., 2012; Gaiero et al., 2013; Timmusk et al., 2017). Medicinal plants, in particular, may select different bacterial communities from the soil through their roots, given that most endophytic bacteria originate in the rhizosphere (Hallmann et al., 1997) or may be inherited through vertical transmission (Santoyo et al., 2016). Promising results in the promotion of plant growth, such as an increase in biomass, the height of the plant, and the depth of the roots, have been obtained through the inoculation of plants or seeds with a large number of endophytic bacteria, including Achromobacter, Microbacterium, Bacillus (Soares et al., 2016), Enterobacter (Khalifa et al., 2016; Castro et al., 2018; Ludueña et al., 2018), Pantoea (Khamwan et al., 2018; Marag and Suman, 2018), Paraburkholderia (Bernabeu et al., 2018), Pseudomonas (Ali et al., 2014), Lactococcus, and Klebsiella (Marag and Suman, 2018). The induction of the production of secondary metabolites by endophytes is one other extremely important aspect of the cultivation of medicinal and aromatic plants, given that they are the source of innumerable biologically-active substances. Some of these metabolites may be produced by a single microorganism or through the integration of this organism with the plant (Brader et al., 2014), vincamine, diosgenin, emodin (Venugopalan and Srivastava, 2015) and taxol (Naik et al., 2019). There is growing interest in these substances as alternative sources for the bioprospection of compounds (Akinsanya et al., 2015), reflecting the potential of endophytic organisms for the synthesis of biotechnological metabolites (Ludwig-Müller, 2015; Palanichamy et al., 2018), which has a number of benefits, such as reduced production costs, structural diversity, and the potential of a range of active compounds for the treatment of different diseases (Alvin et al., 2014; Nisa et al., 2015). The growing interest in the various bioactive properties of A. vera (Bajpai, 2018; Gao et al., 2018) has stimulated further research into the use of endophytic bacteria associated with this plant species as inoculants and elicitors of secondary metabolites,

83 which can contribute to an increase in the plant’s biometric parameters and aloin content, although little is known of the endophytic organisms that promote growth in medicinal plants (Hassan, 2017). Few data are available on the influence of microorganisms in A. vera plants, on the promotion of growth or the production of aloin (Gupta et al., 2012; Sharma et al., 2014). This research has created a range of potential biotechnological applications for both farming and industry. We investigated the effects of the application of endophytic bacteria with potential for the promotion of growth in Aloe vera and the improvement of the production of aloin.

2. Material and methods

2.1. Bacterial strains The four bacterial strains tested in the present study were originally isolated from the roots of Aloe vera growing in three distinct environments (Silva et al., 2019). At least one isolate was selected from each environment, with the 35V (Enterobacter ludwigii) and 135V (Enterobacter tabaci) strains being obtained from the nursery environment, 149H (Paraburkholderia sp.) from a market garden, where there was contact with a range of other herbaceous plants, in particular, medicinal species, and 389C (Pantoea cypripedii) from a natural environment, an open area of anthropogenic habitat with reduced vegetation cover, in the vicinity of the town of Rio Verde, in Goiás, Brazil. The strains have multiple functions capable of promoting plant growth, in particular the production of IAA, in addition to solubilizing Bayóvar phosphates, calcium, and iron, and presenting antibiosis against Sclerotinia sclerotiorum and Fusarium sp (Silva et al., 2019).

2.2. Preparation of the inoculants Each isolate was cultivated in nutrient broth at ambient temperature in a table- type agitator (150 rpm), for 24 h. The final concentration of the cultures was standardized to 109 CFU mL-1. Each plant was either not inoculated (control) or inoculated with one strain or a consortium of the four strains. The consortium was based on a combination of all four cultures, of equal cellular density, mixed in an Erlenmeyer.

2.3. Experiment in pots The experiment was conducted between September 2017 and May 2018. The pots contained a sterilized, sieved soil:sand (2:1) substrate, which was dried for 12 days. This soil consisted of samples of red dystroferric latosol collected from an area of native Cerrado (17°48’28” S, 50º53’57” W, 720 m a.s.l.), at a depth of 10–40 cm. The −3 chemical composition of this soil was pH in CaCl2 = 4.4; P = 1.1 mg dm ; K = 76.7 mg −3 −3 −1 dm ; Ca = 0.47 cmolc dm ; organic matter = 31.8 g∙kg , and base saturation = 11%. The 6 kg plastic pots were filled with approximately 4 kg of this substrate. The A. vera seedlings, with 3–5 leaves and 10-15 cm in height were transplanted to the pots, which were initially keep in greenhouse for 40 days, for adaptation. After 40 days, the plants were inoculated with 5 mL of the bacteria (109 CFU mL-1) at the base of the plants, which were then kept in a greenhouse before being transferred to the open air environment. The experiment had a fully randomized design, with six treatments: control; 35V - Enterobacter ludwigii; 135V - Enterobacter tabaci; 149H - Paraburkholderia sp.; 389C - Pantoea cypripedii; MIX - mixture of the four bacteria, with 10 repetitions. The plants were irrigated whenever necessary. During the experiment, the fungicide Carbendazim (2 mL L-1) was applied five times, and Piraclostrobina; Epixiconazol, three times, for the control of fungal disease in the aerial part of the plant.

2.4. Evaluation of plant growth characteristics Six months after inoculation, four plants from each treatment were removed carefully from the pots for biometric evaluation. Two of the largest leaves of each plant were collected by cutting them off at the base, and then washed under running water. These leaves were standardized by fresh weight and size, to 10 cm from the base of the plant, with all the other leaves being used for the extraction of the gel and latex. The roots were washed and photographed for the analysis of their surface area, diameter and volume, using the SAFIRA software (Jorge and Silva, 2010).

2.5. Determination of the aloin content The leaves of A. vera were washed under running water and then rinsed with distilled water, before being cut at the base to drain off the latex, which was collected over the subsequent two hours. The gel (parenchymal tissue) was separated

85 mechanically from the leaves. The samples were then stored in flasks wrapped in aluminium foil to impede the decomposition of the aloin, frozen, and then lyophilized. The samples of the latex and gel were analyzed individually to quantify the concentration of aloin. The extracts were obtained from 60 mg of latex and 100 mg of the lyophilized gel, extracted in 5 mL of methanol. All the samples were sonicated for 10 minutes and filtered through cottonwool and a Whatman PP 0.45 µm nylon membrane, prior to analysis. A high efficiency Shimadzu® liquid chromatograph, equipped with an LC-20AT quaternary pump system, CTO-20A column oven, SIL-20AHT auto-injector, and DAD SPD-M20A detector, was used to analyze the samples. The chromatographic data were ® ® analyzed in the LC-Solution program. The samples were injected into a Shimadzu C18 Shim Pack VP-ODS (150 x 4.6 mm; 5 μm) chromatographic column coupled to a Shimadzu® ODS pre-column (10 mm x 4.6 mm; 5.0 μm). The chromatographic conditions were as follows: mobile phase A (ultrapure water + 0.1% of acetic acid); mobile phase B (acetonitrile), gradient mode: (Min/A%:B%): 0/80:20; 11.5/50:50; 13/20:80; 19/80:20, with an oven temperature of 40°C; mobile phase flow of 1.0 mL.min-1, detection at 254 nm, and injection volume of 20 µL. The latex samples were diluted (1:10) for injection, while the gel samples were injected without being diluted. The linearity of the method was verified based on the calibration curve. Five different solutions were prepared with different concentrations of aloin (standard aloin, from Aloe barbadensis Miller leaves, ≥ 97% Sigma-Aldrich, CAS 1415-73-2, -1 C21H22O9) (25; 50; 125; 250 and 500 µg mL in methanol from a stock solution of 2.5 mg mL-1). The compound was identified by comparing the retention time with that of the standard compound. The quantity of aloin produced by the plant was calculated based on the sample peak, concentration, and the area of the standard peak, with five repetitions. The retention time of the aloin was 9.17 min (Fig. 1). The aloin was identified by comparison with the standard value, and by using characteristic spectra, obtained using the DAD detector. The method was linear between 25 and 500 µg mL-1 for the aloin content present in the latex and gel samples. The linearity was expressed by the coefficient of determination (r = 0.997) and the equation obtained by the analysis was y=13536x – 35310. The relative standard deviations between the injections and the standard aloin were less than 5% (between 0.37 and 0.68).

Fig. 1. Chromatogram of the standard aloin, extracted from Aloe vera, with 125 μg.mL- 1 and the UV absorption spectrum

2.6. Statistical analysis The biometric data were submitted to the Shapiro-Wilk normality test and then to the analysis of variance (ANOVA). The means were compared by the Dunnett test using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). For aloin content, means were compared by Tukey test (5%) using SISVAR software (Ferreira, 2011). The graphics were built by Sigmaplot 12.0 software (Systat Software Inc.).

3. Results

3.1. Biometric analyses Treatments with plant growth promoting endophytic bacteria, applied individually as inoculants, positively influenced A. vera plants after six months of inoculation. From the Dunnett test, the Control and MIX treatments were compared (Fig. 2). For the total leaf number, fresh leaf weight and fresh and dry root weight parameters, the treatment that provided the best results was the 149H Paraburkholderia sp. Isolate, which increased the number of leaves by 25.9%, fresh weight leaves at 51.8% and fresh and dry root weight (107 and 6.25%), respectively, compared to Control plants and the mixture of all isolates (MIX). No difference was detected between the treatments by evaluating the shoot length and leaf dry weight. For root

87 volume, 135V Enterobacter tabaci treatment provided lower means compared to Control. For root surface area, treatments 149H Paraburkholderia sp. and 389C Pantoea cypripedii did not differ from the Control treatments and were superior to the MIX treatment, along with the other treatments. Root diameter was better with 35V E. ludwigii treatment, with a 33.3% increase compared to the Control.

Fig. 2. Biometric parameters of the Aloe vera plants inoculated with different strains of endophytic bacteria. Uppercase letters compare with the Control treatment and

89 lowercase letters compared with the MIX treatment. (A) Length (cm) of the shoot plant; (B) Number of leaves; (C) Fresh weight (g) of the leaves and root; (D) Dry weight (g) of the leaves and root; (E) Root volume (mm3); (F) Surface area (mm2) of the root, and (G) Diameter of the root (mm). Control = not inoculated; 35V = Enterobacter ludwigii; 135V = Enterobacter tabaci; 149H = Paraburkholderia sp.; 389C = Pantoea cypripedii; MIX = consortium of the four bacteria. In each graph, pairs of columns with different letters at the top have significantly different means (Dunnett) (mean of four replicates)

3.2. Aloin content The A. vera plants inoculated with some of the micro-organisms produced larger quantities of aloin in the dry gel and latex in comparison with the control (Fig. 3). Inoculation with the endophytic bacterium 35V - Enterobacter ludwigii resulted in an increase of 38.8% in the aloin content of the dry gel, in comparison with the other treatments. No major variation was found among most treatments in the case of the dry latex (Fig. 3).

Fig. 3. Aloin content of Aloe vera plants inoculated with different strains of endophytic bacteria: (A) Dry gel; B) Dry latex. Control = not inoculated; 35V = Enterobacter ludwigii; 135V = Enterobacter tabaci; 149H = Paraburkholderia sp.; 389C = Pantoea cypripedii; MIX = consortium of the four bacteria. In each graph, pairs of columns with different letters at the top have significantly different means (Tukey, 5%)

4. Discussion The three bacterial genera used in this work are commonly associated with plants and have a diversity of plant growth promoting species, being them:

Paraburkholderia (Bolívar-Anillo et al., 2016; Huo et al., 2018; Zhou et al., 2018 ), Enterobacter (Andrés-Barrão et al., 2017; Castro et al., 2018) and Pantoea (Lopes et al., 2016; Trifi et al., 2017). A set of mechanisms, attributed by the multifunctional characteristics of endophytic bacteria previously evaluated, such as IAA production, phosphate solubilization and phytopathogen suppression, contributed to the promotion of A. vera growth and increased aloin content. The chemical communication between endophytes and the interior of plant tissues is of high relevance, as such microbiota can produce and secrete low concentrations of metabolites within the plant and exert greater effect on it, compared to those secreted by the rhizospheric microbiota, which is influenced by biotic and abiotic factors, which can neutralize and minimize their effect (Orozco-Mosqueda et al., 2018). Treatment with 149H Paraburkholderia sp. stood out among the others by the increase of the shoot and the gel content. However, regarding the aloin content, different responses were obtained, with a 19.2% increase in dry gel and no effect on dry latex compared to the Control treatment (Fig. 3). Bacteria of the genus Paraburkholderia are described in the literature with nitrogen fixation (N) abilities (De Meyer et al., 2018), phosphate solubilization and antifungal activity (Barnabeu et al., 2018) capable of colonizing the rhizosphere and endosphere, possess a diverse genome and house a wide range of physiological functions. This promotes growth, vitality in various plant species and maximizes tolerance to abiotic and biotic stresses (Mitter et al., 2013). In this work, this can be proved by the increase of biomass production. Some biofunctions exerted by bacteria favors plant nutrition through the fixation of N, solubilization of immobilized phosphates in the soil, and production of phytohormones, such as IAA, which alter root morphology and increase soil water and nutrient absorption (Castanheira et al., 2017) and, consequently, plant growth is favored (Liu et al., 2016). Auxin has roles related to changes in plant division, extension and cell differentiation (Spaepen et al., 2007). This increases root development and xylem, as well as biosynthesis of various metabolites (Glick, 2012), factors that contributed to the increase of A. vera plant shoots and aloin production. Different concentrations of IAA affect plant physiology in different ways, which may vary for each plant species, with some plants being more and others less sensitive, depending on the type of tissue involved, that is, the roots or the shoots (Glick, 2012). In this work, the stimulation caused by IAA in A. vera plants, possibly had greater

91 effect on shoots resulting in increase of biomass and leaf area, which favors the production of photoassimilates for the plant. The growth promoting effect caused by bacteria by phytostimulation with the production of IAA, according to Spaepen and Vanderleyden (2011), occurs through multiple mechanisms, such as N fixation, phosphate solubilization and ACC deaminase activity and results in the promotion of plant growth. and increased yield. The biostimulatory effect caused by phosphorus (P) solubilization favors plant growth, particularly in shoot length (Khamwan et al., 2018). Beneficial bacteria, artificially introduced into the plant, allow access to essential nutrients such as P, which are fundamental for plant growth and development (Ingle and Padole, 2017). While abundant in the soil, in both organic and inorganic forms, P is poorly mobile and its availability is very low to plants, becoming insoluble and unavailable to plants (Behera et al., 2014). This is because phosphate anions participate in reactions that limit their availability by forming compounds such as tricalcium phosphate, iron phosphate or aluminum phosphate, dissolved or crystalline salts, or salts adsorbed by soil colloids (Lobo et al., 2019). Cardarelli et al. (2013) in a study with arbuscular mycorrhizal fungi (AMF) and organic fertilization associated with saline irrigation, observed effect on fresh leaf yield, increased aloin content, β-polysaccharide and improvement of plant phytochemical profile. Sharma et al. (2014), in a study with A. vera micropropagated plants colonized by the symbiotic fungus Piriformospora indica, observed an increase in gel content of 16.5% under in vitro conditions. In this work, a 51.8% increase in leaf fresh content was possible using the bacterium 149H Paraburkholderia sp. Gupta et al. (2012) with the inoculation of A. vera rhizospheric bacteria in the plant itself, also obtained satisfactory results, with the increase of biometric parameters and aloin content, being the best results obtained by the consortium between strains of Pseudomonas synxantha, Burkholderia gladioli, Enterobacter hormaechei and Serratia marcescens. In contrast, in this work, in the consortium treatment (MIX), by mixing the four bacteria: 35V Enterobacter ludwigii, 135V Enterobacter tabaci, 149H Paraburkholderia sp. and 389C Pantoea cypripedii, no satisfactory results were observed. The MIX treatment reflected the results obtained in the plant, in which lower values were found for parameters such as leaf number, fresh leaf and root weight, surface area, root diameter and aloin content in gel and latex. Further studies on the

92 interaction of these strains may help clarify this issue and select compatible strains for best results. According to Sánchez-Machado et al. (2017b), latex contains a higher amount of aloin compared to gel. Such results were observed in the present study. The results of the study indicate that it is possible to achieve higher productivity of A. vera, with faster growth and quality, specifically with an increase of up to 51.8% of biomass and 38.8% of aloin gel content, with the inoculation of 149H Paraburkholderia sp and 35V Enterobacter ludwigii bacteria, respectively. Inoculated strains provided a positive effect on the shoots of A. vera plants compared to the root system, which makes the use of such microorganisms interesting, since the greater number of leaves and gel content are profitable for farmers and advantageous for the industry, as well as an increase in aloin content. A new formulation combination with the bacteria 149H Paraburkholderia sp. and 35V E. ludwigii is suggested for further evaluations under greenhouse conditions, aiming at results of biomass increment and aloin content. The understanding of the interactions between plants and microorganisms provides important insights cor the selection of endophytes in the search for the greatest concentrations of the target metabolites and the best productivity, which will provide larger amounts of raw material for the manufacture of natural medicinal products. To satisfy the demand for medicinal plants from the manufacturing industry, the large-scale multiplication of output, based on both rapid growth rates and higher productivity, is required, and this may be achieved, in part, by the application of multifunctional microorganisms. Over the past decade, a number of studies have shown that, under adequate supervision, although not necessarily applicable to commercial cultivars, the inoculation of plants with growth-promoting microorganisms may have positive results (Bashan et al., 2014). However, much more experimental fieldwork is necessary to optimize the production of inoculants for the market that will guarantee a systematic increase in the productivity and quality of crops, with costs that are accessible to producers. In this way it will be possible to implement smarter agriculture, to implement advantageous environmental technology capable of increasing the yield of plants and phytochemicals of strategic interest.

5. Conclusions Endophytic bacteria obtained from the roots of Aloe vera contributed to the maximization of the growth and biomass of the plant, and may be applied as inoculants for the establishment of a more sustainable for farming system. The bacterium 149H Paraburkholderia sp. was capable of increasing the growth of the aerial portion of the A. vera plant, as well as its production of gel, and the aloin content in this gel. The bacterium 35V Enterobacter ludwigii contributed to an increase in the concentration of aloin in both the gel and latex of A. vera plants.

Acknowledgements The authors thank the Instituto Federal Goiano - Campus Rio Verde for providing the infrastructure and materials to carry out this work.

Conflict of interests The authors declare no conflicts of interest.

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CONCLUSÃO GERAL

Este estudo permitiu conhecer a multifuncionalidade de bactérias radiculares endofíticas cultiváveis de Aloe vera, destacando-se entre estas os isolados 135V e 348V Enterobacter tabaci e 149H Paraburkholderia sp. que expressaram um conjunto de traços funcionais com aplicação para a promoção de crescimento vegetal. Foram selecionados isolados de cada ambiente: 35V Enterobacter ludwigii e 135V Enterobacter tabaci do ambiente viveiro, 149H Paraburkholderia sp. da horta e 389C Pantoea cypripedii campo e utilizadas como inoculantes nas plantas de A. vera. Após seis meses de condução do experimento em vasos, pode-se evidenciar que há endofíticos bacterianos obtidos de suas raízes com papel relevante para melhorar seu crescimento e rendimento, podendo ser usados como inoculantes para estabelecer um sistema de produção agrícola sustentável e produtivo.

A bactéria 149H Paraburkholderia sp. foi capaz de aumentar o crescimento da parte aérea e o conteúdo de gel, além elevar o teor de aloína no gel. A bactéria 35V Enterobacter ludwigii possibilitou um incremento no teor de aloína em gel e látex em plantas de A. vera. O estudo sugere futuras avaliações em nível de campo para a comprovação do potencial dessas estirpes para a utilização como inoculantes, biofertilizantes ou controle biológico de fitopatógenos a fim de substituir produtos químicos e aumentar o crescimento, sanidade e a produtividade de culturas.

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