Quality of Brachiaria Seeds Used in Integrated Systems

FEDERAL UNIVERSITY OF MATO GROSSO

FACULTY OF AGRONOMY AND ZOOTECHNY

Postgraduate Program in Tropical Agriculture

QUALITY OF BRACHIARIA SEEDS USED IN INTEGRATED SYSTEMS

ANNE CAROLINE DALLABRIDA AVELINO

CUIABÁ - MT

2021 2

FEDERAL UNIVERSITY OF MATO GROSSO

FACULTY OF AGRONOMY AND ZOOTECHNY

Postgraduate Program in Tropical Agriculture

QUALITY OF BRACHIARIA SEEDS USED IN INTEGRATED SYSTEMS

ANNE CAROLINE DALLABRIDA AVELINO

Agricultural Engineer

Advisor: PROF. DR. JOADIL GONÇALVES DE ABREU

Co-Advisor: PROF. DR. GIOVANI DE OLIVEIRA ARIEIRA (in memoriam)

A thesis presented to the Faculty of Agronomy and Zootechny of the Federal University of Mato Grosso (UFMT), in fulfilment of the requirements for the degree of Doctor in Tropical Agriculture.

CUIABÁ - MT

2021 3

“Entrega o teu caminho ao Senhor; confia Nele, e o mais Ele fará”.

Salmos 37:5 6

ACKNOWLEDGEMENTS To God, for the gift of life. I thank Thee for being with me in every step, for staying in command, bringing support and blessing. I feel grateful for having received strength and courage in the hard moments, and enough joy to appreciate happiness. Thank Thee, Father, for blooming in my heart the perseverance and the certainty that the obstacles are not greater than Thy might. To my parents, José Neto and Bernardete Dallabrida, for their teaching. I thank their support, comprehension, advising, and warnings, for I am sure that these were uplifting. Blessed is the lord for having me handed to them both, and them to me. Furthermore, I am grateful to my entire family, especially to my sister, Emanuelle, for the companionship. To Prof. Dr. Joadil Abreu, for his advising and for being an example of ethics and professionalism, besides all his patience, dedication and support. To Prof. Dr. Giovani Arieira (in memoriam), for his co-advising and for accompanying and making the experiment feasible. I will be always grateful for the wonderful moments and rich teachings and remember with joy the wonderful person and professional that I had the honor to meet. To Professors Dr. Daniela T. da Silva Campos, Dr. Virgínia Helena de Azevedo, and Dr. Elisangela Clarete Camili for ceeding the space in the laboratories for which they are responsible. To the remaining members of my examination board, Lívia de Barros and Cárita Arantes, for devoting some of their time to help in the improvement of this work. To the “colleagues” of the Department of Zootechny and Rural Extension of the UFMT, for aggregating value to my professional and personal life, especially to Prof. Dr. Nicolau Elias Neto, for being an amazing human being and a competent professional. To Sidnéa Caldeira, for her help and companionship, and for the great gift of her friendship. Thank you for having welcomed, taught and helped me in the laboratory techniques of seed analysis. To Mrs. Noraci Morales, angel of God, for always knowing the right moment to offer a friendly hug. To the remaining professionals that compose the staff of the UFMT, which were in part of my journey in this institution, my eternal gratitude, in a special manner to 7

Professors Dr. Aluísio Brigido Borba Filho, Dr. Leimi Kobayasti, and Dr. Patrícia Helena de Azevedo. To the students that compose the research group in Forage Farming and Phytopathology of the UFMT, campus Cuiabá, which helped in the execution of the experiment. To my advisees and “pupils” Alexandre, Bruno Terzi, Bruno Valiati, Fernando, Jéssica, Lucas Dias, Mariana, Matheus, Onassis, Rodolfo, Victor, Wesley, and Yuri, “auntie loves you”! To my friends, closer than brothers, André, Arthur, Bruna, Dan, David, Dayana, Deyvison, Diego, Dryelle, Elizângela, “Farias Family”, Ianna, Isadora, Jairo, Lucas, Tallita, and Vanessa, for they constitute a family chosen by heart. To the PPGAT and CAPES, the financing agency of the scholarship. I shall never be able to express in words the dimension of my gratitude and happiness. God bless us! Thank you!

QUALITY OF BRACHIARIA SEEDS USED IN INTEGRATION SYSTEMS

ABSTRACT (general): The crop-livestock integration system (ILP) is a model of sustainable cultivation that allows the recovery of degraded pastures and the intensification of pasture and grain production. However, the presence of pathogens in the production fields has hampered the use of these systems. Based on this, the objective was to carry out a bibliographic review describing the main forages and agricultural crops used in crop-livestock integration systems in tropical regions, the main phytonmatodes associated with these crops and the possible control measures, in addition to identifying the associated fungi the main agricultural and forage crops that cause damage to integrated production systems in Brazilian tropical regions and possible control measures. For this, it was based on a bibliographic review of the Scielo, Scopus and Google Scholar databases, with data from 1999 to 2019. Based on the literature, the cultivation of forages belonging to the genera Brachiaria syn. Urochloa sp. and Panicum syn. Megathyrsus sp., together with soybean, corn, sorghum and millet crops are prevalent in the ILP system of tropical regions. The phytomatomatoides Meloidogyne sp., Pratylenchus brachyurus, Heterodera glycines, Rotylenculus reniformis, Aphelenchoides sp., Ditylenchus sp. and Filenchus sp. can cause greater damage to the main cultures used in ILP. No method of control can eradicate phytonmatodes. The most effective control for the studied phytomatodes is through the integrated management of pests and diseases. The main fungi reported as associated with forage and agricultural crops, and soils from integrated systems in tropical Brazilian regions are Bipolaris sp., Curvularia sp., Exserohilum syn. Helminthosporium sp., Phoma sp., Fusarium sp., Macrophomina sp., Pythium sp., Rhizoctonia sp. and Sclerotium sp. The main methods indicated for fungi control are the use of quality seeds, crop rotation, resistant cultivars and chemical treatment of the seeds. Integrated production systems have been developed to preserve productive resources and maintain the profitability of agribusiness. However, the use of seeds of low physiological and sanitary quality for the implantation of agricultural and forage crops in production fields can favor the dissemination and proliferation of phytopathogens. Thus, the objective was to evaluate the effects of the contact times of brachiaria seeds with the chemical fertilizer on the sanitary quality of brachiaria seeds; to identify the main genera of fungi and phytonmatoids associated with the main tropical forage seeds used in integrated systems, and to distinguish the seeds with high physiological and sanitary quality. Three experiments were carried out in a completely randomized design, in the laboratory, using seeds of brachiaria seeds. The first experiment was carried out in a 5x2 factorial scheme, with four replications. The treatments consisted of the contact times (0, 24, 48, 72 and 96 hours) of the fertilizer 05-25-15 (N-P2O5-K2O) with the seeds of Brachiaria syn. Urochoa ruziziensis cv. Kennedy and B. syn. Urochoa brizantha cv. Marandu. After the time of contact of the fertilizer with the brachiaria seeds, the seeds were separated and the analyzes were carried out: water content, germination, health and accelerated aging, with subsequent germination test and sanitary analysis of the seeds. The other two experiments (experiment 1 corresponding to the 2017/18 crop and experiment 2, corresponding to the 2018/19 crop) were performed with four treatments and four replicates each. The treatments consisted of the seeds of Brachiaria syn. Urochoa ruziziensis cv. Kennedy (ruziziensis grasses) and B. syn. Urochoa 9

brizantha cultivars Marandu, Xaraés and BRS Piatã (Marandu, Xaraés and Piatã grasses). Cladosporium sp., Fusarium sp., Rhizoctonia sp., Meloidogyne sp., Filenchus sp., Aphelenchus sp., Aphelencoides sp. and Rotylenchulus sp. are the main phytopathogens found in association with the seeds studied in this work. Comparing the seeds studied in this work, the seeds of ruziziensis grasses have better physiological quality and the seeds of xaraés grasses have better sanitary quality.

Keywords: contact, fertilizer, fungi, germination, phytonematodes, tropical grasses, vigor.

QUALIDADE DE SEMENTES DE BRAQUIÁRIA UTILIZADAS EM SISTEMAS DE INTEGRAÇÃO

RESUMO (geral): O sistema de integração lavoura-pecuária (ILP) é um modelo de cultivo sustentável que permite a recuperação de pastagens degradadas e a intensificação da produção de pasto e grãos. No entanto, a presença de patógenos nos campos de produção tem dificultado o emprego desses sistemas. Com base nisso, objetivou-se realizar uma revisão bibliográfica descrevendo as principais forrageiras e culturas agrícolas utilizadas em sistemas de integração lavoura-pecuária em regiões tropicais, os principais fitonematóides associados a essas culturas e as possíveis medidas de controle, além de identificar os fungos associados às principais culturas agrícolas e forrageiras que ocasionam danos aos sistemas integrados de produção de regiões tropicais brasileiras e as possíveis medidas de controle. Para isso, baseou-se em uma revisão bibliográfica das bases de dados Scielo, Scopus e Google Scholar, com dados de 1999 a 2019. Com base na literatura, o cultivo de forragens pertencentes aos gêneros Brachiaria syn. Urochloa sp. e Panicum syn. Megathyrsus sp., juntamente com as culturas da soja, milho, sorgo e milheto são predominantes no sistema ILP das regiões tropicais. Os fitonematoides Meloidogyne sp., Pratylenchus brachyurus, Heterodera glycines, Rotylenculus reniformis, Aphelenchoides sp., Ditylenchus sp. e Filenchus sp. podem causar maiores danos às principais culturas utilizadas em ILP. Nenhum método de controle pode erradicar os fitonematóides. O controle mais efetivo para os fitonematóides estudados é através do manejo integrado de pragas e doenças. Os principais fungos relatados como associados às culturas forrageiras e agrícolas, e solos de sistemas integrados de regiões tropicais brasileiras são Bipolaris sp., Curvularia sp., Exserohilum syn. Helminthosporium sp., Phoma sp., Fusarium sp., Macrophomina sp., Pythium sp., Rhizoctonia sp. e Sclerotium sp. Os principais métodos indicados para controle de fungos são o uso de sementes de qualidade, rotação de culturas, cultivares resistentes e tratamento químico das sementes. Sistemas integrados de produção foram desenvolvidos para preservar os recursos produtivos e manter a rentabilidade do agronegócio. Contudo, o uso de sementes de baixa qualidade fisiológica e sanitária para a implantação de culturas agrícolas e forrageiras em campos de produção pode favorecer a disseminação e proliferação de fitopatógenos. Sendo assim, objetivou-se avaliar os efeitos dos tempos de contato de sementes de braquiária com o fertilizante químico na qualidade sanitária das sementes de braquiária; identificar 10

os principais gêneros de fungos e fitonematoides associados às principais sementes forrageiras tropicais utilizadas em sistemas integrados, e distinguir as sementes com elevada qualidade fisiológica e sanitária. Foram realizados três experimentos em delineamento inteiramente casualizado, em laboratório, utilizando sementes de sementes de braquiária. O primeiro experimento foi realizado em esquema fatorial 5x2, com quatro repetições. Os tratamentos consistiram nos tempos de contato (0, 24, 48, 72 e 96 horas) do fertilizante 05-25-15 (N-P2O5-K2O) com as sementes de Brachiaria syn. Urochoa ruziziensis cv. Kennedy e B. syn. Urochoa brizantha cv. Marandu. Decorridos os tempos de contato do fertilizante com as sementes de braquiária, procedeu-se a separação das sementes e realizaram-se as análises: teor de água, germinação, sanidade e envelhecimento acelerado, com posterior teste de germinação e análise sanitária das sementes. Os outros dois experimentos (experimento 1 correspondendo a safra 2017/18 e experimento 2, correspondendo a safra 2018/19) foram realizados com quatro tratamentos e quatro repetições cada. Os tratamentos consistiram nas sementes de Brachiaria syn. Urochoa ruziziensis cv. Kennedy (capim-ruziziensis) e B. syn. Urochoa brizantha cultivares Marandu, Xaraés e BRS Piatã (capins Marandu, Xaraés e Piatã). Cladosporium sp., Fusarium sp., Rhizoctonia sp., Meloidogyne sp., Filenchus sp., Aphelenchus sp., Aphelencoides sp. e Rotylenchulus sp. são os principais fitopatógenos encontrados em associação às sementes estudadas neste trabalho. Comparando as sementes estudadas neste trabalho, as sementes de capim-ruziziensis possuem melhor qualidade fisiológica e as sementes de capim-xaraés possuem melhor qualidade sanitária.

Palavras-chave: capins tropicais, contato, fertilizante, fungos, germinação, fitonematoides, vigor

TABLE OF CONTENTS

Page 1. Introduction (General) ...... 12 1.1 References (General) ...... 15 2. Review: Phytonematodes in Integrated Crop-Livestock Systems of Tropical Regions ...... 18 3. Review: Fungi Associated with Major Agricultural and Forage Crops in Integrated Systems of Brazilian Tropical Regions ...... 43 4. Effect of Fertilizer on Palisadegrass Seeds Pathogens ...... 66 5. Impact of Phytopathogens on the Seed Quality of Tropical Grasses in Integrated Systems...... 81 6. Conclusions (General) ...... 103 APPENDIX ...... 104

1. INTRODUCTION (General) The presence of degraded pastures is particularly common in agricultural frontier areas in the country, such as the North and Midwest regions. In these places, this phenomenon is directly associated with the low productivity of livestock and the increase in deforestation. Several sources indicate that, of the 172 million hectares of pastures in Brazil, more than 60% are in some stage of degradation. In Cerrado areas, which account for 60% of the country's meat production, around 80% of the 45-50 million hectares with cultivated pastures show some degree of degradation (Nogueira, 201-?), Crop-livestock integration has been pointed out as the alternative to promote a socioeconomic and sustainable development given the growing demand for food and the need for reducing the opening of new areas, increasing the efficiency of the production system (Almeida, 2015; Cordeiro et al., 2015; Balbino et al., 2011; Vilela et al., 2011). With the sustainable increase in Brazilian livestock production, comparing the data obtained in 2010 with the information from 2018, an improvement in the quality of pastures was observed in the evaluated period, with a reduction from 29.1% to 41.1% in the percentage of degraded areas (Ferreira Júnior, et. al, 2020). This techonology is based on different productive systems of grains, fibers, meat, milk, and agroenergy through rotation, intercropping and/or succession of cattle-raising and agricultural activities in the same area (Alvarenga and Noce, 2005; Macedo, 2009; Vilela et al., 2008). Its objectives are the rational maximization of the use of natural resources, the diversification of production, and the reduction of the economic risks and farming production costs, besides contributing to the recovery of degraded areas (Leonel, 2011; Macedo, 2009; Teixeira et al., 2018; Vilela et al., 2011; Zonta et al., 2016). In this system, the most used forage plants were introduced from Africa and belong to the genera Brachiaria syn Urochloa, Panicum syn. Megathyrsus sp., and Andropogon (Macedo and Zimmer, 2007). The introduction of these forage plants provided a considerable increase in the animal filling of pastures (Foloni, et al., 2009) and made cattle raising viable in poor and acid soils, which are predominant in Brazil (Souza et al., 2006). 13

However, the reduced size of the seeds of forage species hardens their isolate and uniform distribution and, consequently, the obtainment of an ideal plant population (Lima et al., 2000). Aiming to avoid this problem, the forage sowing can be performed simultaneously in the sowing line of the agricultural crop. In that regard, the forage seeds can be mixed to the fertilizer at the crop establishment, stored in the compartment of the broadcast spreader and distributed in the same depth as the fertilizer (Duarte and Maria, 2013; Kluthcouski et al., 2000). In this sowing model, due to the contact of the seeds of forage species with the chemical fertilizer, the seeds are subjected to unfavorable factors, such as the saline and phytotoxic effects of the fertilizers (Dan et al., 2011; Lima et al., 2009; 2010; 2013; Peres et al., 2012; Rezende et al., 2012). In spite of the reports regarding the negative impacts of the prolongation of the contact time on the physiological quality of seeds, there is a scarcity of information related to the impacts of the contact with the fertilizer on the sanitary quality of the seeds. Another obstacle to the adoption of integrated production systems are the phytopathogens associated to the forage seeds, since these cause deleterious effects in the seeds and in the development and persistence of the plants in the area (Lucca, 1985; Mallmann et al., 2013; Marchi et al., 2007; 2010; Sbalcheiro et al., 2014; Santos et al., 2014) As a consequence of the expansion of the cultivated area, especially of Brachiaria syn Urochloa, there was an increase in the incidence of phytopathogens (fungi and phytonematodes) in the production fields. This fact is probably derived from the introduction and dissemination of pathogens to areas free of contamination (Cardoso et al., 2014; Lopes et. al, 2009). Furthermore, the absence of a sanitary quality standard for the forage Grass seeds commercialized in the national market, allied to the inadequate management of integrated production systems (Marchi et al, 2007; Vechiato e Aparecido, 2008) also contributes for the increase in the incidence of phytopathogens in producing areas. Given the economic and environmental damages caused by the phytopathogens, it is necessary to draw efficient control strategies. Toward that purpose, it is first necessary to identify the phytopathogens. 14

Based on this, this research aimed to evaluate the effects of the contact time of Brachiaria seeds with the chemical fertilizer on the sanitary quality of the seeds, as well as to identify the main genera of fungi and phytonematodes associated to the primary forage seeds used in integrated systems of tropical regions, distinguishing the seeds with high physiological na sanitary quality.

1.1 REFERENCES (General) Almeida RG, Ramos AKB, Zimmer AH, Machado LAZ, Kichel NA, Gontijo Neto MM et al. Performance of tropical forages in crop-livestock and crop-livestock-forest integration systems. In: Cordeiro LAM, Vilela L, Kluthcouski J, Marchão RL, editors. Crop-livestock- forest integration: the producer asks, Embrapa responds. 1st ed. Brasília: Embrapa, 2015. Portuguese.

Alvarenga RC, Noce MA. Crop and livestock integration. Sete Lagoas: Embrapa, 2005.

Balbino LC, Barcellos AO, Stone LF. Reference document: crop-livestock-forestry integration. 1st ed. Brasília: Embrapa; 2011. Portuguese.

Cardoso ED, De Sá ME, Haga KI, Binotti FFS, Nogueira DC, Valério Filho WV. Physiological and performance in overcoming dormancy in seeds Brachiaria brizantha under artificial chemical treatment and aging. Semina: Ciências Agrárias. 2014;35:21- 38.

Cordeiro LAM, Vilela L, Marchão RL, Kluthcouski j, Martha Junior GB. Integrated crop- livestock and integrated crop-livestock-forestry systems: strategies for sustainable intensification of soil use. Cadernos de Ciência & Tecnologia. 2015;32(½):15-43. Portuguese.

Dan HA, Dan LGM, Barroso ALL, Braccini AL, Puccinin GG. Mixture of seeds of Brachiaria ruziziensis G.et e with urea aiming at the implantation of the crop-livestock integration system. Revista Caatinga. 2011;24(4):68-73. English.

Duarte AP, Maria IC. Maize + brachiaria: minimum investment, maximum return. A Lavoura, 2013;697:39-43.

Ferreira Júnior, LG, Oliveira-Santos C, Mesquita VV, Parente L.L. Dynamics of Brazilian pastures: Occupation of areas and signs of degradation - 2010 a 2018. Lapig: UFG, 2020. 19p. Portuguese.

Foloni JSS, Custódio CC, Caldeira FJ, Calvo CL. Emergence of seedlings of Brachiaria brizantha influenced by seed scarification, fertilizer use and seeding depth. Científica, 2009;37:89 - 97.

Kluthcouski J, Cobucci T, Aidar H, Yokoyama LP, Oliveira IP, Costa JLS, Silva JG, Vilela L, Bacellos AO, Magnabosco CU. Santa Fé system: crop-livestock integration by the consortium of annual crops with forages, in crop areas, in the direct and conventional systems. Santo Antonio de Goiás: Embrapa, 2000.

Leonel FP. Crop-Livestock integration: production and quality of forage. In: Zervoudakis JT, Cabral LS. Nutrition and production of beef cattle. Cuiabá; 2011:221-233. Portuguese.

Lima EDV, Cavariani C, Lima P, Crusciol C, Nakagawa J, Villas Boas R. Physiological quality of millet seeds (Panicum dichotomiflorum Mix) as a function of mixing time with triple superphosphate. Cultura Agronômica. 2000;9(1):177-189. Portuguese.

Lima EDV, Tavares JC, Azevedo VR, Leitão-Lima PS. Mixture of Brachiaria brizantha seeds with NPK fertilizer. Rural Science. 2010;40(2):471-474. English. DOI: 10.1590 / S0103-84782010005000003.

Lima EDV, Tavares JC, Silva EC, Leitão-Lima P. Triple superphosphate as route of distribution of Brachiaria brizantha seeds for renewal of pastures in the Amazon. Revista Brasileira de Zootecnia. 2009;38(5):796-800. English. DOI: 10.1590 / S1516- 35982009000500003.

Lima EV, Tavares JCS, Leitão-Lima PS, Pinheiro DP. Contact periods of the KCl fertilizer on the physiological quality of Brachiaria brizantha Stapf seeds. Revista Amazônia Ciência e Desenvolvimento. 2013;38(5):53-64. English.

Lopes J, Fortes CA, Souza RM, Tavares VB. Importance of seed quality for pasture establishment. Pubvet, 2009;3.

Lucca O. The Importance of Sanitation in High Quality Seed Production. Revista Brasileira de Sementes, 1985;7:113-123.

Macedo MCM, Zimmer AH. Integrated crop and livestock systems in the Cerrado region of Brazil. In: International Symposium on Crop-Livestock Integration. Annals. Curitiba: UFPR, UFRGS, Ohio State University; 2007.

Macedo MCM. Crop and livestock integration: the state of the art and the near future. Brazilian Journal of Animal Science. 2009;28:133‑146.

Mallmann G, Verzignassi JR, Fernandes CD, Santos JM, Vechiato MH, Inácio CA, Batista MV, Queiroz CA. Fungi and nematodes associated with tropical forage seeds. Summa Phytopathologica. 2013;39:201-203.

Marchi CE, Fernandes CD, Bueno ML, Batista MV, Fabris LR. Fungi associated to commercial seed of braquiaria grass. Arquivo Instituto Biológico. 2010;77:65-73.

Marchi CE, Fernandes CD, Borges CT, Santos JM, Jerbal VF, Trentin RA, Guimaraes LRA. Phytopathogenic nematofauna of commercial seeds of tropical forages. Pesquisa Agropecuária Brasileira. 2007;42:655-660.

regi%C3%B5es%20Norte%20e%20Centro%20Oeste.&text=Diversas%20fontes%20indi cam%20que%2C%20dos,em%20algum%20est%C3%A1gio%20de%20degrada%C3% A7%C3%A3o. Accessed April 20, 2021. Portuguese.

Peres AR, Vazquez GH, Cardoso RD. Physiological potential of Brachiaria brizantha cv. Marandu seeds kept in contact with phosphatic fertilizers. Brazilian Journal of Seeds. 2012;34(3):424-432. Portuguese. DOI: 10.1590 / S0101-31222012000300009.

Rezende AV, Andrade LP, Almeida GBS, Rabelo CHS, Rabelo FHS, Landgraf PRC, Nogueira DA, Vilela HH. Effect of depth and seed mix on chemical fertilizer on emergence of seedlings of forage species. Revista Agrarian, 2012;5:115-122.

Santos GR, Tschoeke PH, Silva LG, Silveira MCAC, Reis HB, Brito DR, Carlos DS. Sanity analysis, transmission and pathogenicity off fungi associated with forage plant seeds in tropical regions of Brazil. Journal of Seed Science, 2014;36:54-62.

Sbalcheiro CC, Jose SCBR, Barbosa JCRCM. Physiological and sanitary quality, and transmission of fungi associated with Brachiaria brizantha (Hochst. ex. A. Rich.) Stapf seeds submitted to thermal and chemical treatments. Journal of Seed Science, 2014;36:443-450.

Souza FHD, Pott EB, Primavesi O, Bernardi ACC, Rodrigues AA. Alternative uses of residual straw from seed production for pasture. São Carlos: Embrapa, 2006.

Teixeira D, Alves DB, Rodrigues G, Bizerra KM, Souza L, Gregório M. The forage plant in the ICL e ICLF. Course work. Bahia: State University of Bahia, 2018.

Vechiato HM, Aparecido CC. Fungi in forage grass seeds: phytosanitary restriction and detection methods. São Paulo: Biological Institute; 2008. Portuguese.

Vilela L, Marta Junior GB, Marchão RL, Guimarães Junior R, Barioni LG, Barcellos AO. Integrated crop-livestock. In: Faleiro FG, Farias Neto AL. Savanas: challenges and strategies for the balance between society, agribusiness and natural resources. Planaltina: Embrapa, 2008.

Vilela L, Martha Junior GB, Macedo MCM, Marchão RL, Guimarães Júnior R, Pulrolnik K et al. Integrated crop-livestock systems in the Cerrado region. Pesquisa agropecuária. 2011; 46(10):1127-1138. Portuguese.

Zonta JH, Sofiatti V, Silva ORRF, Ramos EM, Barbosa HF, Cordeiro Junior AF, Lira AJS. Integrated crop-livestock systems (ICLS) for the Agreste region of the Northeast. Campina Grande: Embrapa, 2016.

2. REVIEW: PHYTONEMATODES IN INTEGRATED CROP-LIVESTOCK SYSTEMS OF TROPICAL REGIONS

ABSTRACT: The integrated crop-livestock system (ICLS) is a model of sustainable cultivation that allows the recovery of degraded pastures and the intensification in pasture and grain production. However, the presence of pathogens in the production fields has hampered the employment of these systems. In order to minimize or eradicate the phytonematodes in the production fields and the seeds used in ICLS, it is necessary to know the nematofauna. Based on this, this work aimed to perform a literature review describing the main forages and agricultural crops used in integrated crop-livestock systems in tropical regions, the major phytonematodes associated with these crops and their control measures. This work was based on a literature review from the Scielo, Scopus and Google Scholar databases, with data from 1999 to 2019. The initially used keywords were "tropical weeds"; "Agricultural crops"; and "ICLS" and their respective terms in the Portuguese language. From the initial results, we used the keywords "Brachiaria syn. Urochloa sp. "; "Phytonematodes"; "Millet"; "Maize"; "Panicum sp." (syn. Megathyrsus); "Soy"; "Sorghum", and "ICLS" and their respective terms in the Portuguese language. Publications that did not meet the criteria of this study (analyzed by titles and abstract) were considered as exclusion criteria, as well as repeated works in the databases. Based on the literature, the cultivation of forages belonging to the genera Brachiaria syn. Urochloa sp. and Panicum syn. Megathyrsus sp., along with and soybean, maize, sorghum and millet are predominant in the ICL system of tropical regions. The phytonematodes Meloidogyne sp., Pratylenchus brachyurus, Heterodera glycines, Rotylenculus reniformis, Aphelenchoides sp., Ditylenchus sp. and Filenchus sp. cause greater severity of damage to the main cultures used in ICL. No control method can eradicate the phytonematodes. The most effective control for the studied phytonematodes is through integrated pest and disease management.

Keywords: Brachiaria syn. Urochloa sp., phytonematode control, maize, Panicum sp.,soybean.

REVISÃO: FITONEMATOIDES EM SISTEMA DE INTEGRAÇÃO LAVOURA- PECUÁRIA DE REGIÕES TROPICAIS

RESUMO: O sistema de integração lavoura-pecuária (ILP) é um modelo de cultivo sustentável que permite a recuperação de pastagens degradadas e a intensificação da produção de pasto e grãos. No entanto, a presença de patógenos nos campos de produção tem dificultado o emprego desses sistemas. Para minimizar ou erradicar os fitonematoides nos campos de produção e nas sementes utilizadas no ILP, é necessário conhecer a nematofauna. Com base nisso, este trabalho teve como objetivo realizar uma revisão bibliográfica descrevendo as principais forrageiras e culturas agrícolas utilizadas em sistemas de integração lavoura-pecuária em regiões tropicais, os principais fitonematóides associados a essas culturas e as possíveis medidas de controle. Este trabalho foi baseado em uma revisão bibliográfica das bases de dados Scielo, Scopus e Google Scholar, com dados de 1999 a 2019. As palavras-chave 19

utilizadas inicialmente foram "tropical weeds"; "Agricultural crops"; e "ICLS" e seus respectivos termos na língua portuguesa. A partir dos resultados iniciais, foram utilizadas as palavras-chave "Brachiaria syn. Urochloa sp."; "Phytonematodes"; "Millet"; "Maize"; "Panicum sp." (syn. Megathyrsus); "Soy"; "Sorghum", and "ICLS" e seus respectivos termos na língua portuguesa. As publicações que não atenderam aos critérios deste estudo (analisadas por títulos e resumos) foram consideradas como critérios de exclusão, bem como trabalhos repetidos nas bases de dados. Com base na literatura, o cultivo de forragens pertencentes aos gêneros Brachiaria syn. Urochloa sp. e Panicum syn. Megathyrsus sp., juntamente com as culturas da soja, milho, sorgo e milheto são predominantes no sistema ILP das regiões tropicais. Os fitonematoides Meloidogyne sp., Pratylenchus brachyurus, Heterodera glycines, Rotylenculus reniformis, Aphelenchoides sp., Ditylenchus sp. e Filenchus sp. podem causar maiores danos às principais culturas utilizadas em ILP. Nenhum método de controle pode erradicar os fitonematóides. O controle mais efetivo para os fitonematóides estudados é através do manejo integrado de pragas e doenças.

Palavras-chave: Brachiaria syn. Urochloa sp., controle de fitonematoides, milho, Panicum sp, soja.

2.1 Introduction The concern with environmental preservation has resulted in restrictions by the current legislation to the opening of new areas for agriculture and exploitation of native wood. Allied to this, there is a growing demand for food due to the increase in the world population, which requires an increase in the agricultural output (Balbino et al., 2011; Vilela et al., 2011). To aggravate the situation, it is estimated that 20% of world pastures are in the process of degradation (United Nations Environment Programe, 2004). The presence of degraded pastures is particularly common in agricultural frontier areas in the country, such as the North and Midwest regions. In these places, this phenomenon is directly associated with the low productivity of livestock and the increase in deforestation. Several sources indicate that, of the 172 million hectares of pastures in Brazil, more than 60% are in some stage of degradation. In Cerrado areas, which account for 60% of the country's meat production, around 80% of the 45-50 million hectares with cultivated pastures show some degree of degradation (Nogueira, 201-?), In the tropical region, pasture degradation is highlighted as a reflection of poor management, which reduces the productive potential of cattle. Therefore, there is a 20

need for the development of agricultural and livestock processes and technologies, aiming at the efficient use of inputs (Dias Filho, 2011; Balbino et al., 2011; Vilela et al., 2011). With the sustainable increase in Brazilian livestock production, comparing the data obtained in 2010 with the information from 2018, an improvement in the quality of pastures was observed in the evaluated period, with a reduction from 29.1% to 41.1% in the percentage of degraded areas (Ferreira Júnior, et. al, 2020). Sustainable crop models were developed in this regard, such as the integrated crop-livestock system (ICL), allowing the recovery or renewal of degraded pastures, the intensification in pasture production, and grain production. The maximization of food production in response to intensified livestock farming rules out the need to open up new areas so that the demands may be met (Cordeiro et al., 2015; Leonel, 2011; Macedo, 2009; Vilela et al., 2011). However, the presence of pathogens in production fields has hampered the implementation of integrated systems since they may contain soils and crops that are susceptible to these pathogens. In addition, the incidence of pathogens has become a problem of growing importance, and it is believed that phytonematodes can influence the forage and the intercropped agricultural crops from the establishment up to harvest stages (Vechiato and Aparecido, 2008; Euclides et al., 2010). Several strategies have been reported in studies in order to minimize the phytonematodes in the production fields and, consequently, in the seeds employed in the agricultural production systems (Favoreto et al., 2006; Garcia et al., 2000; Garcia and Tenente, 2001; Tenente, 2003). However, it is necessary to know the nematofauna in the production fields of the main forages and agricultural crops, so that corrective actions may be applied in order to reduce or eradicate the incidence of phytonematodes in the production fields of integrated crop-livestock systems. Based on the foregoing, a literature review was performed to describe the main forages and agricultural crops used in integrated crop-livestock systems in tropical regions, as well as the main phytonematodes associated with these crops and their possible control measures. 21

2.2 Methodology This work was based on a literature review from the Scielo, Scopus and Google Scholar databases, with data between 1999 and 2019. The initially used keywords were “capins tropicais”; “culturas agrícolas”; and “ILP”, and their respective English terms “tropical grasses”; “agricultural crops”; and “ICL”. At that moment, the purpose was to describe which forages and agricultural crops were the most used in integrated crop-livestock systems in tropical regions. For the inclusion criteria, publications (papers, books, theses, dissertations and scientific communications) released within 1999 and 2019, in the Portuguese and English languages, which fit the purpose of describing the main forages and agricultural crops used in tropical regions were employed in the research. Publications prior to 1999 that did not fit the study aim (analyzed by titles and abstract) were considered as exclusion criteria, as well as works that were repeated in the databases. Based on the results, the second stage of the study was carried out, dealing with the description of the phytonematodes associated with these main crops. Therefore, the keywords employed were “fitonematoides”; “Brachiaria syn. Urochloa sp.”; “Panicum sp.” (syn Megathyrsus); “soja”; “milho”; “sorgo”, “milheto”; “controle” and “ILP” and their respective English terms "phytonematodes"; “Brachiaria syn. Urochloa sp.”; “Panicum sp.” (syn. Megathyrsus); „”soybean”; “maize”; “sorghum”; “millet”; “control” and “ICL”; under different combinations. After reading the titles of the papers and other publications, 97 works that met the initially proposed criteria were selected and read in full.

2.3 Results and Discussion 2.3.1 Main Forages and Agricultural Crops in ICL of Tropical Regions The widely used forage grasses in the ICL systems of tropical regions belong to the genera Brachiaria syn. Urochloa and Panicum syn. Megathyrsus sp. In the case of B. syn. Urochloa, the species B. syn. Urochloa ruziziensis and some cultivars within the species B. syn. Urochloa brizantha are highlighted, such as Marandu, Xaraés and Piatã (Embrapa Sede, 201-?. a; Euclides et al., 2005; Semeata; Valle et al., 2004). 22

In general, forage grasses of the genus B. syn. Urochloa are widely employed in integrated crop-livestock systems in tropical regions, as they may present drought, fire and shade tolerance, pest resistance, along with high levels of productivity, nutritional value, and regrowth capacity. In addition, there is a need for well-drained soils, from average to high fertility (Embrapa Sede, 201-?; Euclides et al., 2005; Semeata; Valle et al., 2004) Regarding the forage of the genus Panicum syn. Megathyrsus sp., the Panicum syn. Megathyrsus maximum cultivars Tanzânia and Mombaça are highlighted. These cultivars have tolerance to grasshoppers, which makes them a good alternative when aiming to implant an ICL system. Despite having high demands on soil fertility and requiring deep and well-drained soils, they successfully adapt in tropical regions (Embrapa Sede, 201-?. b; Embrapa Sede, , 201-?. c; Pedreira et al., 2006) The main agricultural crops are soybean and maize, followed by sorghum and millet, with the main factor behind the great exploitation of these cultures being economical, along with the sowing period, specific agricultural defenses and tolerance to water stress (Alvarenga et al., 2007; 2011; Balbino Júnior, et al., 2009; Costa et al., 2008; Pedreira et al., 2006; Priesnitz et al., 2011; Rodrigues and Cruz, 2009). Soybean has been used in rotation with other grain crops and forage grasses, since it minimizes the use of inputs for being a nitrogen-fixing plant, also favoring the recovery of degraded areas. The intercropping with grasses is difficult, but there are indications that the application of narrow leaf herbicide subdoses helps to reduce the loss of grain yield in broadleaf crops (such as soybean), at the same time allowing the grasses to settle (Balbino Júnior et al., 2009; Pedreira et al., 2006). Maize is a widely employed crop as it can be used for human consumption and animal feed by marketing all grain or surplus production. Its management presents a diversity of post-emergence narrow-leaf herbicides that are selective to maize, facilitating the intercropping with grasses in ICL systems. Moreover, it is the most frequently employed alternative in soybean succession in integrated systems of tropical regions, maintaining production levels (Alvarenga et al., 2011; Brandt et al., 2006; Cruz et al., 2011; Guimarães et al., 2003). 23

Sorghum and millet have an excellent ability to withstand environmental stresses, maintaining yield in water deficit and low soil fertility conditions. In addition, they have shown to be an excellent alternative to be used in succession to summer crops in ILC systems of tropical regions, especially soybean. Besides, these crops are used for soil covering and straw formation in systems of minimum cultivation or no-tillage, in the case of millet (Alvarenga et al., 2007; Costa et al., 2008; Priesnitz et al., 2011; Rodrigues and Cruz, 2009). Due to the aforementioned expressiveness of the referred crops in integrated crop-livestock systems, they were selected for the analysis of the nematofauna from the seeds and from the production system.

2.3.2 Main Phytonematodes Associated with Major Forages and Agricultural Crops in ICL of Tropical Regions There may be a reduction in the plant stand and development when the association of pathogens with forage seeds occurs (B. syn. Urochloa brizantha and Panicum syn. Megathyrsus maximum), as well as in the persistence of the forages in the area, seed production, dry mass and nutritional value, adversely affecting the production of products such as milk and meat. It is also noted that the presence of the pathogen in the seeds constitutes a barrier to commercialization (Favoreto, 2004; Mallmann et al., 2013). The main phytonematodes found in association with seeds of tropical forage grasses are Aphelenchoides spp. and Ditylenchus spp. (Marchi et al., 2007). The seeds of Panicum syn. Megathyrsus maximum may be infected with Aphelenchoides spp. and Ditylenchus spp. (Mallmann et al., 2013); whereas the seeds of B. syn. Urochloa brizantha may be associated with Aphelenchoides sp., Ditylenchus spp., Filenchus sp., Meloidogyne sp., Pratylenchus brachyurus, Heterodera glycines and Rotylenchulus reniformis (Dias et al., 2010; Favoreto, 2004; Mallmann et al., 2013). Although Ditylenchus spp. phytonematodes tend to be associated with seeds and impurities (soil, straw, and so forth.) of commercial lots of P. syn Megathyrsus maximum, and Ditylenchus spp. and Filenchus sp. to seeds and impurities of commercial lots of B. syn. Urochloa brizantha, only secondary damages have been verified to the B. syn. 24

Urochloa brizantha, P. syn Megathyrsus maximum, and the main agricultural crops (intercropped, succession and/or rotation) used in the ICL system in tropical regions (Marchi et al., 2007). In the main agricultural crops used in the ILC, the phytonematodes that can be propagated via seeds and are the most expressive in terms of affecting productivity are Meloidogyne sp., Pratylenchus sp., Heterodera sp., and Rotylenchulus sp. (Goulart, 2008; Inomoto, 2011; Marchi et al., 2000; Silva et al., 2017). Regarding the phytonematodes and their damage to agricultural crops, soybean is the most negatively affected cultivation due to the allied farming system, the agricultural operations that provide proper conditions for the development of these pathogens. Thus, certain phytonematodes have become one of the primary phytosanitary problems of the soybean and maize crops, being able to drastically reduce grain yields and even turn farming areas unfeasible (Dias et al., 2010).

2.3.3 Aphelenchoides sp. Studies that relate to the pathogenicity of the genus Aphelenchoides sp to B. syn. Urochloa brizantha are scarce and inconclusive. The phytonematode Aphelenchoides besseyi, despite being found in association with P. syn Megathursus maximum, causes secondary damage to this forage (Dias et al., 2010; Garcia et al., 2000). However, studies have related the incidence of A. besseyi to the appearance and severity of the Mad Soy II disease (MS-II), which causes green stem and leaf retention in the soybean crop, also being able to attack cotton crops (Dias et al., 2010; Favoreto et al., 2015; Meyer et al., 2017). This disease is a serious problem in Brazil, affecting soybean and cotton yields, with estimated yield reductions from 60 to 100% due to a high index of abortion of flowers and pods and loss of grain quality, along with the wrinkling and darkening of leaves (Dias et al., 2010; Embrapa sede, 201-? b, c; Meyer et al., 2017; Favoreto et al., 2017). The presence in the seeds and production fields of pathogenic fungi such as Fusarium graminearum, Fusarium pallidoroseum, Alternaria, Sclerotinia, Phomopsis 25

spp, Rhizoctonia, Phytium inflatum and Phytophphora sojae is another problem to be considered, since they act as multipliers of Aphelenchoides sp. (Lopes et al., 2018). The literature suggests that the adopted cultural management can help to reduce the incidence of Mad Soy II. The practice of cultural control can be adopted through post-harvest weed control measures, such as the early desiccation at sowing, the use of crop rotation or resistant cultivars, properly considering that Crotalaria juncea, C. spectabilis, C. ochroleuca, maize, millet, sorghum, and rice are not multipliers of these phytonematodes. B. syn. Urochloa brizantha and B. syn. Urochloa ruziziensis, however, present a low or no final population of A. besseyi (Favoreto et al., 2018; Meyer et al., 2015; Silva et al., 2018a; 2018b).

2.3.4 Meloidogyne sp. Phytonematodes of the genus Meloidogyne present several hosts, causing damage to numerous crops (coffee, cotton, soybean, maize, etc.), with greater impact and importance on soybean and maize (Dias et al., 2010; Freitas et al., 2001). Furthermore, they have a wide geographical distribution, with M. incognita and M. javanica being the main species for tropical regions. M. incognita predominates in areas of coffee and cotton farming, whereas M. javanica occurs in a generalized way (Dias et al., 2010; Freitas et al., 2001). The damages caused by these phytopathogens are the intense abortion of pods after flowering, chlorotic spots or necrosis between the veins, small and yellowish plants in reeds, besides the early maturation of the plants. In addition, with the occurrence of the "Indian summer" phenomenon, the damage might be more severe (Embrapa Soja, 2010; 2013). However, in the roots of the plants infected by the pathogen, the main characteristic of this parasitism is the formation of galls or tumors. Galls have their number and size dependent on the susceptibility of the cultivar and on the population density of the pathogen (Embrapa Soja, 2010; 2013). With regard to nematode incidence control methods, studies indicate that the forages Brachiaria syn. Urochloa brizantha and P. syn Megathyrsus maximum may favor 26

phytonutrient suppression by M. incognita and M. javanica (Dias-Arieira et al., 2003; Galbieri et al., 2015). Furthermore, it is verified that the use of genetic resistance associated with biological and chemical control provides an increase in the green mass production of the shoot part of the soybean crop in soils infested with Meloidogyne spp. (Araújo et al., 2012). Biological control with the fungi Paecilomyces lilacinus (Thom.) and P. chlamydosporia may contribute to the control of M. incognita (Jacobs et al., 2003; Cadioli et al., 2009; Nunes et al., 2010). With regard to chemicals with nematicidal activity, we highlight avermectin and oxime methylcarbamate, used via seed treatment in soybean. They act by suppressing the phytopathogen in the initial development stage of the crop (Monfort et al., 2006; Cabrera et al., 2009; Kubo et al., 2012; Araújo, 2013). Moreover, the use of organic acids such as humic and fulvic acids may control the incidence of M. incognita and M. javanica in crops such as sugarcane (Gaviglia, 2013). Based on the chemical treatment of phytonematodes, the use of abamectin via seeds has shown to be promising for M. incognita in the cotton crop (Bessi et al., 2010), as well as and via soil in the control of Meloidogyne javanica (Corte et al., 2014). However, it is worth noting that the aforementioned control methods did not promote the eradication of phytonematodes in the area, but only a reduction in its incidence. Therefore, there may occur failure of the integrated systems with agricultural crops (soybean, maize, etc.) and susceptible forages in areas of Meloidogyne sp., as the remaining parasites could reproduce and develop, increasing the population index and causing severe damage to the susceptible crops implanted in the productive system (Dias et al., 2010).

2.3.5 Pratylenchus brachyurus Another nematode that causes economic losses in tropical regions, especially in crops such as soybean, rice, sugarcane, sorghum, cotton, beans, tropical grasses, and especially maize, is Pratylenchus brachyurus (root lesion nematode) (Asmus, 2004; Goulart, 2008). 27

The damages caused by the root lesion nematode are dependent on the susceptibility of the crop, the population level within the productive system, the environmental condition and the type of soil. In addition, the level of severity and the appearance of some symptoms from the attacks of this nematode are associated with conditions of water stress, compacted and mainly sandy soils, conditions in which the pathogen can cause significant damage to production even in a low population density (Dias et al., 2010; Perry et al., 2009). There was a reduction of 30% in the production of infected soybean in field experiments, and 90% damage in common bean infected with P. brachyurus. However, in tropical regions, this severity can compromise up to 50% of the soybean production from infested areas (Goulart, 2008; Machado et al., 2007). In production fields from the state of Mato Grosso do Sul, Brazil, there are frequency reports of 82, 79 and 87% in the cities of Chapadão do Sul, Costa Rica, and São Gabriel do Oeste, respectively (Amus, 2004; Ribeiro et al., 2014), causing a loss of 30% in soybean yield (Dias et al., 2010). The attacks and the manifestation of symptoms can occur in defined areas, easily visualized in the field. The parasitized plants present reduced growth but remain green, being in a stage of partial or total obscuration during the penetration of cortical and corticoid cells, instead of neutralizing the toxins during the feeding process. In addition, the movement of the parasites through the root is disorganized, aiding in the destruction of cells (Dias et al., 2010; Melo et al., 2012). Furthermore, root lesions caused by the nematode serve as an entry point for bacteria and fungi, primarily the wilt-causing fungi Fusarium and Verticillium, causing necrosis and rot in the plant (Dias et al., 2010; Grigolli et al., 2014). With regard to the possibility of P. brachyurus suppression, studies have found that B. syn. Urochloa brizantha and B. syn. Urochloa ruziziensis allowed the population increase of phytonematodes of different origins (okra (Pb20) and cotton roots (Pb24)). However, the Pb24 cotton showed a higher proliferation in areas cultivated with the forages studied in this work, as well as with soybean and maize cultivars (Hirakuri et al., 2012; Inomoto and Machado, 2007). 28

This suggests that this nematode may be severely reduced in integration systems considering succession, rotation or consortium utilizing tropical grasses, soybean, maize and cotton (Hirakuri et al., 2012; Inomoto and Machado, 2007). A viable alternative is the use of rotating crops with species that are not hosts and susceptible to the pathogen, such as Crotalaria spectabilis and C. breviflora, which present P. brachyurus reproduction factors of approximately 0.1 and 0.2%, respectively (Hirakuri et al., 2012; Inomoto and Machado, 2007; Inomoto et al., 2006; Inomoto, 2008; Motta et al., 2005). In addition to the use of Crotalaria spectabilis and C. breviflora, other non- multiplying species for P. brachyurus can be used in integrated systems, such as C. ochroleuca, C. juncea, black oat (Avena strigosa Schreb), amaranth (Amaranthus cruentus), and the intercropped of C. spectabilis and millet (Pennisetum glaucum). The result is a reduction in the population density of this phytonematode in the productive systems (Asmus and Inomoto, 2016; Debiasi et al., 2016; Inomoto et al., 2006). Furthermore, the employment of soybean cultivars with a low P. brachyurus reproduction factor can be used as a palliative method in high incidence areas, such as BRS GO Chapadões; Conquista; MG/BR 46; M-SOY 8378; M-SOY 8360RR; M-SOY 8800; UFUS Guará; and UFUS 37 (Ribeiro et al., 2007; Silva, 2014). Another palliative alternative is the biological control through the fungus Pochonia chlamydosporia (Costa, 2015), and the chemical control with abamectin and imidacloprid with thiodicarb via seed treatment (Bortolini et al., 2013). As previously explained, it should be noted that the aforementioned control methods did not promote the eradication of phytonematodes in the area, but only a reduction in their incidence. This will generate the same complications already discussed.

2.3.6 Heterodera glycines Another phytonematode that causes economic losses in the soybean crop is the Heterodera glycines (soybean cyst nematode). This nematode penetrates the roots of the plant, hinders the absorption of nutrients and water and prevents the development of 29

the plant. Due to this, the plant can manifest small size and chlorosis in the shoot part (Dias et al., 2010; Henning et al., 2014). This nematode penetrates the roots of the soybean plant and impedes the absorption of water and nutrients, which results in reduced size and chlorosis in the shoot part; these are known as yellow dwarfism. However, in regions with more fertile soils and good rainfall distribution, the absence of symptoms in the shoot part of the plant is common. This shows that the definitive diagnosis always requires the observation of the root system (Dias et al., 2010; Henning et al., 2014). Generally, root-related symptoms of infected plants begin 30 to 40 days after soybean sowing. The root system is reduced in this period, exhibiting female nematodes. After the death of this female, the body is transformed into a hard structure of dark brown color, called cyst, that detaches itself from the root and is allocated into the soil (Freitas et al., 2001; Embrapa Soja, 2010; 2013). The dissemination of the cyst occurs primarily due to the transporting of the infected soil; therefore, poorly harvested seeds, implements, and machinery, as well as winds and water can disseminate the nematode into new areas. In properties where the ICL system is employed, it is very important to know the origin of the forage seeds, as it may contain the inoculum of the cyst nematode (Dias et al., 2010). In areas with the presence of H. glycines, the adoption of joint control measures (integrated management) is a more effective strategy (Arantes et al., 1999; Garcia et al., 1999). Biological control is a promising option to be used in conjunction with cultural control (crop rotation), soil management and the use of resistant cultivars (Embrapa, Soja, 2002; Hewlet et al., 2007; Pedroso et al., 1999; Santos, 2016). Biological control with the fungus Pasteuria lilacinus associated with chemical control, as well as the use of abamectin along with Pasteuria nishizawae and Pasteuria metchnikoff, in isolated ways, are effective options to control H. glycines in the soybean crop (Almeida et al., 2018; Neves et al., 2009; Santos, 2016). The control via seeds is the most effective and viable way to apply such treatments (Hewlet et al., 2007; Pedroso et al., 1999). With regard to the crop control and the resistance of cultivars, it is seen that millet is resistant to Heterodera glycines (Inomoto and Asmus, 2013). Due to their efficiency 30

and economic viability, the use of resistant cultivars is the most adopted control method for H. glycines (Niblack et al., 2002).

2.3.7 Rotylenchulus reniformis There are reports that the phytonematode Rotylenchulus reniformis (reniform nematode) has only caused severe damage to cotton. However, after the 1990s, there were also attacks on soybean crops. It is believed that this phytonematode occurs in high population densities and is present in 29% of soybean-cultivated areas in the state of Mato Grosso do Sul, Brazil (Embrapa Soja, 2010; 2013). The symptoms of nematode attack in soybean plants resemble those caused by problems of soil compaction and mineral deficiency, with the presence of unevenness in the crops, extensive areas of underdeveloped plants characterized by dwarfism, chlorosis, and foliar yellowing. There is no formation of galls; however, in some points of the root, it is possible to observe an outer layer of soil adhered to the egg mass of the nematode (Dias et al., 2010; Asmus and Ishimi, 2009). Sexually immature reniform females cause attacks and subsequent manifestation of symptoms. These migrate in the soil in search of the roots of the host plant and penetrate them until reaching the region before the pericyclic cells, initiating the parasitism. Upon reaching sexual maturity, the outer portion of the root system acquires a resemblance similar to that of a kidney, giving rise to the name "reniform nematode" (Asmus and Ishimi, 2009; Santos, 2019). The control of Rotylenchulus reniformis can be performed with the cultural method and resistance of cultivars, by cultivating sorghum and millet. In addition to the mentioned species, soybean cultivars are being studied with regard to the resistance mechanism to this phytonematode, with promising present results (Inomoto and Asmus, 2013; Mello et al., 2013). However, long periods of rotation with resistant crops are necessary, since this parasite can survive for up to two years in the soil due to the mechanism of anhydrobiosis (Asmus and Inomoto, 2007; Lira and Moura, 2013). In general, for some crops, there is still the recommendation of chemical control for all treated phytonematodes. These control substances are known as nematicides 31

and can be applied via soil or seed. However, their effectiveness and economic viability are still poorly understood (Guarnieri, 2018), varying according to the form of application and the chemical group employed (Corte et al., 2014). It is believed that the joint application via seed and soil has a greater residual effect in the control of phytonematodes (Corte et al., 2014). Several nematicides of the carbamate and organophosphorus chemical groups are available for seed treatment in Brazil (Ribeiro et al., 2014).

2.3.8 Phytonematodes Eradication In general, the currently used phytometric control methods are preventive control (to avoid entry and dissemination in the production field), crop control (rotation and/or succession of resistant and host crops), genetic control (resistant cultivars), chemical and biological control (Guarnieri, 2018). Phytonematode control through different methods is important for reducing the damage caused by the parasites (Araujo et al., 2012). It is always preferable to prevent the spread of the nematode than to treat an infested area (Ferraz et al., 2010), since they are difficult to eradicate from the production field. Furthermore, the occurrence of multiple phytonematode species in one area may be a control problem, since different strategies can be adopted (Macsorley, 2002). There are several tested techniques for the eradication of phytonematodes present in forage seeds. In the P. syn Megathyrsus maximum seed the treatment of the seeds at 60 °C for 10 minutes or at 57 °C for 15 minutes eradicated the seed phytonematodes without compromising seed quality (Garcia et al., 2000). In addition, the use of irradiation has been promising as an effective method for the eradication of seed pathogens. This is due to high control efficiency without toxic residues and no side effects (Garcia et al., 2000). However, it is emphasized that the above-mentioned technique was only used in laboratory experiments and with a small amounts of seeds. In order for this method to be used in commercial forage seeds, it will be necessary to develop techniques that allow large-scale eradication and with no any side effects on the seeds Favoreto, 2008). 32

Briefly, based on the scientific findings presented in this paper, the following table was generated (Table 1). 33

Table 1. Most occurring regions, susceptible crops, damages and control of the phytonematodes discussed in this paper. Phytonematodes Susceptible crops Damages Controls Aphelenchoides sp. P. syn Megathursus In plants: reductions from 60 Cultural control with: Crotalaria maximum, soybean and cotton. to 100% due to a high index of juncea, C. spectabilis, C. ochroleuca, abortion of flowers and pods and maize, millet, sorghum, rice, B. syn. Urochloa brizantha and B. syn. Urochloa loss of grain quality, along with the ruziziensis. wrinkling and darkening of leaves Meloidogyne sp. Coffee, cotton, soybean, In plants: intense abortion of Control cultural: Brachiaria syn. maize. pods after flowering, chlorotic spots Urochloa brizantha and P. syn or necrosis between the veins, small Megathyrsus maximum and yellowish plants in reeds, besides the early maturation of the plants Pratylenchus Soybean, rice, In plants: reduced growth but Control cultural: rotation or brachyurus sugarcane, sorghum, cotton, remain green; the movement of the consortium utilizing tropical grasses (B. beans, tropical grasses, and parasites through the root is syn. Urochloa brizantha and B. syn. especially maize. disorganized, aiding in the Urochloa ruziziensis), soybean, maize and destruction of cells. cotton Heterodera glycines Soybean. In plants: reduced size and Biological control with the fungus chlorosis in the shoot part; root Pasteuria lilacinus associated with system reduced, exhibiting female chemical control; cultural control (crop nematodes. After the death of this rotation), soil management and the use of female, the body is transformed into resistant cultivars; crop control and the a hard structure of dark brown color, resistance of cultivars: millet is resistant called cyst. Rotylenchulus Cotton; soybean. In plants: dwarfism, Cultural method and resistance of reniformis chlorosis, and foliar yellowing; no cultivars, by cultivating sorghum and millet. galls.

2.4 Conclusion The cultivation of forages belonging to the genus Brachiaria syn. Urochloa and Panicum syn. Megathyrsus, as well as soybean, maize, sorghum and millet are predominant in the ICL system of tropical regions. The phytonematodes Meloidogyne sp., Pratylenchus brachyurus, Heterodera glycines, Rotylenchulus reniformis, Aphelenchoides sp., Ditylenchus sp. and Filenchus sp cause greater damage to the main crops used in ICL of tropical regions. No control method can eradicate the phytonematodes. The most effective control of the studied phytonematodes is through integrated pest and disease management.

2.5 References Almeida JÁ, Souza CJ, Araújo FG. Seed treatment with abamectin and Paecilomyces lilacinus in the management of Heterodera glycines in soybean. Multi-Science Journal. 2018;1(4):62-65. Portuguese. DOI: 10.33837/msj.v1i4.127.

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3. REVIEW: FUNGI ASSOCIATED WITH MAJOR AGRICULTURAL AND FORAGE CROPS IN INTEGRATED SYSTEMS OF BRAZILIAN TROPICAL REGIONS

ABSTRACT: Integrated production systems were developed to preserve productive resources and maintain the profitability of agribusiness. However, the use of seeds of low physiological and sanitary quality and the implantation of agricultural and forage crops in production fields of low sanitary quality may favor the dissemination and proliferation of phytopathogens such as fungi. Therefore, using the scientific literature, this work aimed to identify the fungi associated with the main agricultural and forage crops that cause damage to the integrated production systems of Brazilian tropical regions and their control measures. This work was based on a literature review in the Scielo, Scopus and Google Scholar databases, with data obtained between 1999 and 2019. The keywords employed were “fungus”, “tropical grass”; “agricultural crops”; “ICLS” (Integrated Crop-Livestock); and “ICLF” (Crop-Livestock-Forest) and their respective terms in Portuguese, under different combinations. For the inclusion criteria, publications (papers, books, theses, dissertations, and scientific communiqués) from 1999 to 2019 which fit the study aim were selected, both in the Portuguese and English languages. The publications that did not meet the criteria of this study and were repeated in databases were considered as exclusion criteria. The main fungi associated with forage and agricultural crops and soils of integrated systems of Brazilian tropical regions are Bipolaris sp., Curvularia sp., Exserohilum syn. Helminthosporium sp., Phoma sp., Fusarium sp., Macrophomina sp., Pythium sp., Rhizoctonia sp. and Sclerotium sp. The main methods of fungal control are the use of quality seeds, crop rotation, resistant cultivars, and chemical seed treatment.

Keywords: Crop rotation, fungicides, resistant cultivars, sustainable agriculture, tropical grass.

REVISÃO: FUNGOS ASSOCIADOS COM AS PRINCIPAIS CULTURAS AGRÍCOLAS E FORRAGEIRAS EM SISTEMA DE INTEGRAÇÃO LAVOURA-PECUÁRIA DE REGIÕES TROPICAIS BRASILEIRAS

RESUMO: Sistemas integrados de produção foram desenvolvidos para preservar os recursos produtivos e manter a rentabilidade do agronegócio. Contudo, o uso de sementes de baixa qualidade fisiológica e sanitária para a implantação de culturas agrícolas e forrageiras em campos de produção pode favorecer a disseminação e proliferação de fitopatógenos, como os fungos. Sendo assim, com consulta à literatura, objetivou-se identificar os fungos associados às principais culturas agrícolas e forrageiras que ocasionam danos aos sistemas integrados de produção de regiões tropicais brasileiras e as possíveis medidas de controle. Esse trabalho foi elaborado a partir de revisão de literatura nas bases de dados Scielo, Scopus e Google Scholar, no período entre 1999 e 2019. As palavras chaves utilizadas foram “fungus”, “tropicals 44

grass”; “agricultural crops”; “ICLS” (Integração lavoura-pecuária); e “ICLF” (Integração lavoura-pecuária-floresta); e seus respectivos termos em português, sob combinações diferenciadas. Para os critérios de inclusão foram selecionadas publicações (artigos, livros, teses, dissertações e comunicados científicos) de 1999 até 2019, nas línguas portuguesa e inglesa, que se enquadrassem no objetivo do estudo. Foram critérios de exclusão as publicações que não preenchiam os critérios deste estudo e que se repetiam nas bases de dados. Os principais fungos relatados como associados às culturas forrageiras e agrícolas, e solos de sistemas integrados de regiões tropicais brasileiras são Bipolaris sp., Curvularia sp., Exserohilum syn. Helminthosporium sp., Phoma sp., Fusarium sp., Macrophomina sp., Pythium sp., Rhizoctonia sp. e Sclerotium sp. Os principais métodos indicados para controle de fungos são o uso de sementes de qualidade, rotação de culturas, cultivares resistentes e tratamento químico das sementes.

Palavras-Chave: agricultura sustentável, capim tropical, cultivares resistentes, fungicidas, rotação de culturas.

3.1 Introduction Agribusiness is worldwide in importance, providing nutrients and contributing significantly to the world economy. Due to the widespread essentiality of agribusiness, there is a need to keep the systems that compose it increasingly productive while preserving the land and reducing the need to open new areas. In order to achieve this purpose, sustainable cultivation models have been created and perfected, such as Integrated Crop-Livestock (ICLS) and Crop-Livestock-Forest (ICLF) systems. These systems aim at the maximum use of the land without degradation, improvement in the physical-chemical quality of the soil, in the zootechnical indexes of the animal component and income diversification (Almeida et al., 2015; Balbino et al., 2011; Costa and Rava, 2003; Leonel, 2011; Macedo, 2009; Mello, 2003; Vilela et al., 2011). Both of the above-mentioned integrated systems (ICLS and ICLF), if poorly managed, might suffer from pest and disease attacks. In addition, the implantation of low sanitary quality crops in production fields, culminating in the use of seeds with low physiological and sanitary quality may favor the dissemination and proliferation of pests and phytopathogens in productive areas, reducing the yield of agricultural and forage crops and, consequently, affecting animal performance (Vechiato and Aparecido, 2008; Vechiato et al., 2010; Fernandes, 2013). 45

Among the phytopathogens that can affect productive areas, fungi and nematodes are the ones that cause most concern as they can decimate crops when in high incidence, being difficult to eradicate from the production system (Fernandes, 2013). In order to adopt fungal control measures, aiming at the maximum yield of integrated systems in Brazilian tropical regions, it is necessary to know the fungal incidence in the production fields of the main crops used in these systems, considering that the cultivation of forages belonging to the genera Brachiaria syn. Urochloa and Panicum syn. Megathyrsus sp., as well as agricultural crops such as soybean, maize, sorghum, and millet, are predominant in integrated production systems (Embrapa Sede a, b, c; Semeata; Pedreira et al., 2006; Alvarenga et al., 2007; 2011; Rodrigues and Cruz, 2009). Based on this, this study aimed to identify the fungi associated with the main agricultural and forage crops that damage the integrated production systems of Brazilian tropical regions, as well as their control measures.

3.2 Methodology This work was based on a literature review in the Scielo, Scopus and Google Scholar databases, with data between 1999 and 2019. The keywords employed were “fungus”, “tropical grass”; “agricultural crops”; “ICLS”; and “ICLF”; and their respective terms in Portuguese “fungos”, “capins tropicais”; “culturas agrícolas”; “iLP”; and “iLPF”, under different combinations. For inclusion criteria, publications (papers, books, theses, dissertations, and scientific communiqués) from 1999 to 2019 which fit the study aim were selected, both in the Portuguese and English languages. Publications prior to 1999 that did not meet the criteria of this study (analyzed by titles and abstract) and were repeated in the databases were considered as exclusion criteria. After reading the titles of articles and other publications, we selected 100 publications that met the initially proposed criteria, which were read in full. 46

3.3 Results and Discussion Fungi are phytopathogenic agents at higher rates of association with seeds, presenting longevity in the productive system through the production of resistance structures (Brasil, 2009; Kruppa and Russomano, 2009). They are among the main causes of diseases in forage plants, causing losses in the yield and quality of the green mass produced, besides reducing the quality (germination and vigor) of the seeds (Lucca-Filho et al., 1999; Mallmann et al., 2013; Marchi et al., 2010; Marcos et al., 2015; Mertz et al., 2007; Schafer and Kotanen, 2003; Vechiato and Aparecido, 2008; Vechiato et al., 2010). Among the fungi present in soils used in integrated systems, and the fungi associated with Brachiaria syn. Urochloa sp., Panicum syn. Megathyrsus sp. and main agricultural crops, there are phytopathogenic taxa belonging to the genera Bipolaris sp., Curvularia sp., Exserohilum syn. Helminthosporium sp., Phoma sp., Fusarium sp., Macrophomina sp., Pythium sp., Rhizoctonia sp., and Sclerotium sp.. There are also secondary fungi such as Alternaria sp., Aspergillus sp., Cladosporium sp., Epicoccum sp., Nigrospora sp., Penicillium sp., and Trichoderma sp. (Athayde Sobrinho and Belmino, 201-?; Embrapa Soja, 2008; Fernandes et al., 2005; Lasca et al., 2004; Mallmann et al., 2013; Marchi et al., 2006; 2007; 2010; Marcos et al., 2015; Silva et al., 2014; Vechiato and Aparecido, 2008; Vechiato, 2004; Vilela Júnior et al., 2011. Witt et al., 2015). Due to the percentage of occurrence and incidence, and the incidence of damage, certain genera of fungi will be described below.

3.3.1 Bipolaris sp. Bipolarys sp. is a pathogenic fungus with a 100% seed transmission rate to the seedlings. It is the main fungus that attacks Panicum syn. Megathyrsus sp., causing leaf spot disease. However, this fungus can be associated with the integument and interior of the seeds and with crop residues of susceptible crops, such as tropical grasses, maize, wheat, rice, and coffee. In addition, it is favored by temperatures between 22 and 30 °C (Amorim et al., 2011; Bueno et al., 2007; Caligiorne et al., 1999; Costa et al., 47

2014; Danelli et al., 2010; Mallmann et al., 2013; Marchi et al., 2007; 2011; Quadros et al., 2012; Santos et al., 2014; Tavanti et al., 2016). The disease and its symptoms are similar in susceptible cultures, being manifested as brown spots and coalescence of the lesions in episodes of severe infestation. These symptoms can be observed 50 days after the sowing of susceptible crops in infected areas and/or using contaminated seeds (Costa et al., 2014; Marchi et al., 2011; Michalski et al., 2007). In addition to the symptoms of contamination by phytopathogens in tropical plants, it causes disturbances in animals and humans, such as allergies, pulmonary and cutaneous infections (Fyen et al., 1999). Listing as alternatives to control the disease, the use of resistant cultivars and fungicides is commonly employed. The application of pyraclostrobin associated with epoxyconazole or tebuconazole alone has been promising for the reduction of the disease intensity in P. syn Megathyrsus maximum seed production fields, increasing the speed of germination. In addition, cultural control, such as crop rotation, is an effective measure for disease control, since it reduces the initial inoculum (Costa et al., 2014; Marchi et al., 2011; Faria et al., 2003). Furthermore, the fungal control can be performed by means of seed treatment with fungicides, aiming to reduce the incidence of fungi in the seeds and in the soil, especially in the initial stages of development of the susceptible plants, besides avoiding the introduction or re-inoculation of phytopathogens (Carvalho and Nakagawa, 2000; Guerra and Costa, 2012).

3.3.2 Curvularia sp. Curvularia sp. is a pathogenic fungus found in several regions of the world that has an important incidence in forage plants of the P. syn Megathyrsus maximum species, causing the leaf spot disease (Mallmann et al., 2013; Marchi et al., 2010; Menten and Ishizuka, 2015). Furthermore, it can cause rotting, reduction of germination up to seed unfeasibility (associating to the integument and endosperm), and death of seedlings in susceptible species, such as tropical grasses and maize, reproducing asexually by ascomycetes (Ferreira, 2010; Guimaraes and Lima, 2019; Lasca et al., 2004; Marchi et al., 2011; Revankar, 2007; Santos et al., 2014; Yago et al., 2011). 48

In addition to the diseases that it causes in susceptible plants, the fungus can cause allergic conditions, endocarditis, pheochycosis, mycetoma, onychomycosis, keratitis, brain abscesses, urinary and pulmonary infections, and infectious wounds in animals and humans (Carter and Boudreaux, 2004; Dasgupta et al., 2005; Herraez et al., 2001; Moura et al., 2001; Pimentel et al., 2005; Shan et al., 2008). For the Curvularia sp. control it is possible to employ a seed treatment with the thiram or thiram-associated carboxin fungicides, which also have efficiency in the control of other fungi, such as Alternaria sp., Gerlachia sp. and Dreschlera sp.; as well as the fungicides fludioxonil combined with metalaxyl-M, colorless fludioxonil comminuted with metalaxyl-M, and thiram alone, which also have fungus control efficiency on Penicillium sp., Alternaria sp., Drechslera sp., and so forth. In addition, the association of thiram and thiabendazole fungicides in seed treatment may promote a greater emergence of seedlings (Lasca et al., 2004; Pinto et al., 1984; Schuch et al., 2006).

3.3.3 Exserohilum syn. Helminthosporium sp. Such as the fungus Curvularia sp., the fungus Exserohilum syn. Helminthosporium sp. causes rotting, seed unfeasibility and death of seedlings, as well as leaf and stem spot diseases. The manifested symptoms are necrotic and elliptic lesions with intense sporulation of the pathogen (Costa et al.; Vechiato et al., 2010; Marchi et al., 2011;) It survives in crop remains (saprophytic form) and in the soil, forming resistance structures and associating to seeds (in tegument and endosperm) and hosts, such as tropical grasses, sorghum and maize (causing helminthosporiosis). In addition, the fungus is favored by temperatures between 18 and 27 °C and high moisture. The main control method is through genetic resistance and crop rotation with resistant cultures (Amorim et al., 2014; Bueno et al., 2007; Costa et al.; Danelli et al., 2012; Quadros et al., 2012; Santos et al., 2014).

3.3.4 Phoma sp. The pathogenic fungus Phoma sp. affects several crops from many continents, such as soybean and coffee. In the European region, the main etiological agent in 49

soybean is Phoma pinodella (=Phoma sojicola). The causal agent in Brazil is the fungus Phyllosticta sojicola (Aveskamp et al., 2008; Irinyi et al., 2008; Salgado and Pfenning, 2000; Vilela Júnior et al., 2011). Furthermore, this fungus also focuses on forages of the genus P. syn Megathyrsus maximum. The symptoms depend on the severity of the incidence, beginning four or five days after the inoculation, causing leaf spot with elongated, necrotic and irregular characteristics, rotting, seed unfeasibility and death of seedlings. By possessing fast and aggressive growth, it can even kill infected seeds before germination (Anjos et al., 2005; Mallmann et al., 2013; Marchi et al., 2011; Menten and Ishizuka, 2015). The fungus belongs to the group of mitosporic fungi and is a Coelomycete, presenting cylindrical, hyaline and small non-septate conidia (Leite et al., 2001). An efficient alternative to control this fungus is the treatment of seeds with thiram fungicides, thiram associated to carboxin, fludioxonil combined with metalaxyl-M, colorless fludioxonil comminuted with metalaxyl-M, and thiram alone (Lasca et al., 2004; Pinto et al., 1984; Schuch et al., 2006).

3.3.5 Fusarium sp. Fungi of the genus Fusarium sp. cause damage to agricultural production systems composed of several agricultural crops, tropical grasses, and animals. This occurs due to the incidence of fusariosis and production of mycotoxin. The symptom of fusariosis in plants is dependent on the phytopathogen species and on the interspecific relation between host-phytopathogen. Mycotoxins can cause symptoms such as false heat, abortion, stillbirths, infertility, problems in the digestive system, bleeding, anemia due to the destruction of the bone marrow, vomiting, necrosis of the epidermis and death of the animals (Carmo, 2017; Nor Azliza et al., 2014; Silva, 2009). The F. solani species causes red root rot in the soybean crop, a symptom of which is the rotting of the root system; F. moniliforme causes the fusarium rot disease in crops of cotton, rice, maize, sorghum, and tropical grasses, in addition to the potential for intoxication of animals due to the production of mycotoxins (zearalenone, fumonisins, and vomitoxins or deoxynivalenol). F. graminearum, F. equiseti, and F. tricintum also 50

produce mycotoxins in maize, sorghum, soybean, wheat, and oat crops, as well as F. pallidoroseum, which can break the stem and lead to the tipping of the cotton, beans and soybean plants (Agrolink, 201-?. a; Silva, 2009). Furthermore, tropical grass seeds susceptible to phytopathogens may increase the inoculum potential in the area and act as a reservoir for future dissemination in crops that will succeed in the area, such as pine, cotton, wheat, rice, bean, soybean, maize, sugarcane, and so forth. The incidence level can be influenced by ideal climatic conditions of high temperature and soil moisture (Athayde Sobrinho and Belmino, 201-?; Mallmann et al., 2013; Marchi et al., 2010) An effective measure for the control of these phytopathogens is the chemical treatment of seeds with fungicides. Among the available options in the commercialization, as previously mentioned for the control of Curvularia sp. and Phoma sp, the use of thiram or carboxin associated with thiram is highlighted, besides the fungicides fludioxonil combined with metalaxyl-M, colorless fludioxonil comminuted with metalaxyl-M, and thiram alone. In the absence of resistant cultivars, well-drained and fertilized soils and healthy and certified seeds can be employed (Athayde Sobrinho and Belmino, 201-?; Lasca et al., 2004; Schuch et al., 2006).

3.3.6 Macrophomina sp. The genus Macrophomina sp. inhabits the soils and manages to multiply in vegetal remains by means of its propagation structures produced by the mycelium (microsclerocios). These structures are resistant to adverse conditions for long periods in the soil. The ideal conditions for phytopathogens to develop are high temperatures and moisture. Moreover, after the insertion into the production area, it attacks roots, stems, leaves, and fruits of susceptible crops, such as sunflower, cotton, sorghum, maize, soybean, and bean, among others. The main species, M. phaseolina, triggers symptoms corresponding to grayish lesions that may evolve to rot and tissue destruction. On the other hand, this tissue disruption causes chlorosis, wilt, drought, and death of susceptible plants (Athayde Sobrinho and Belmino, 201-?; Batista et al., 2016). The broad spectrum of susceptible species and the absence of resistant cultivars hinder the control through crop rotation, although the performing of this practice with 51

forage grasses is recommended. Also, it is always recommended to use healthy and certified seeds (Athayde Sobrinho and Belmino, 201-?). It is observed that soybean plants produce the phytoalexin gliceolin when infected by M. phaseolina, a compound that has the potential to restrict the development of the mentioned fungus through the rapid biosynthesis of glycerol by the plant during fungal infection. This feature may promote genetic improvement programs in the search for resistant plants to M. phaseolina. As a palliative measure to reduce the incidence of these phytopathogens, it is possible to use cultivars with higher tolerance to drought and/or high temperatures (Almeida et al., 2014; Lygin et al., 2010).

3.3.7 Pythium sp. This pathogen is an inhabitant of the soil which can infect seeds and seedlings. It can be associated with plant remains (saprophytes) or susceptible plants, such as soybean, sorghum, cotton, bean, maize, wheat and tropical grasses. The fungus presents resistance structures (oospores) that allow its survival in adverse conditions (Agrolink, 201-?. a;b). The most frequent species are P. graminicola and P. debaryanum, but P. ultimum causes the most impact. Due, the lesions develop rapidly causing chlorosis, growth reduction, wilt, root rot and tipping of the plant in ideal conditions of development (high moisture and mild temperature) (Agrolink, a; b). Furthermore, it also causes economic losses to the agricultural production system since it contributes to the onset of sudden death (root rot and plant tipping) of the Marandu grass (B. syn. Urochloa brizantha cv. Marandu), along with Rhizoctonia sp., Fusarium sp. and water stress (Andrade and Valentim, 2007; Marchi et al., 2011).

3.3.8 Rhizoctonia sp. The fungus Rhizoctonia is a saprophytic fungus, being able to exert parasitism on several crops and animals. It presents a high gene flow, genetic diversity, sexual reproduction and dispersion of clones with high adaptability, as well as producing resistance structures (sclerotia) which remain in the soil for long periods (Agrolink, 201- ?. a; Dias et al., 2019; Poloni et al., 2016). 52

The R. solani species can cause leaf burning, collection rot and death in tropical grasses, soybean and maize; in soybean, it causes plant tipping and root rot, which may reduce the vigor and germination rate of the seeds, as well as toxins that inhibit plant growth. In cotton, the fungus causes the tipping of the plants (Agrolink, 201-?. a; Dias et al., 2019; Goulart, 2016; Poloni et al., 2016). The methods that have effectiveness in controlling the fungi living in the soil, such as Pythium sp. and Rhizoctonia sp., are based on the chemical treatment of seeds, rotation of crops with resistant species (grasses) and elimination of crop residues. With regard to the biological control, isolates of the fungus Trichoderma spp. have effectiveness in the control of R. solani (Athayde Sobrinho and Belmino, 201-?; Lewis and Lumsden, 2001; Luccon et al., 2009).

3.3.10 Sclerotium sp. The main species, Sclerotium rolfsii, lives in the soil and affects crops of soybean, bean, potato, and tomato, among others. It causes the rotting of roots and colon, wilt and tipping of infected plants. The symptoms are manifested in the region of the lap of the plant and correspond to dark spots that originate the cortical rot. This rot can be identified by the formation of a white mycelium and brown-colored resistance structures (sclerocytes). The destruction of tissues occurs under these structures and, with that, wilt, drought and death of the plants. The ideal development conditions occur in regions of tropical climate, with temperatures within 25 and 35 °C and soil moisture in 70% of the field capacity (Athayde Sobrinho and Belmino, 201-?; Agrolink, 201-?. a; Pinheiro et al., 2010). As a control method, the need to use healthy and certified seeds, the elimination of crop residues and crop rotation with maize and cotton (resistant plants) are highlighted, as well as the efficiency of the fungicide tebuconazole in the colony growth and in the germination of sclerotia of S. rolfsi with regard to the chemical control (Athayde Sobrinho and Belmino, 201-?; Cilliers et al., 2003; Prahbu and Hiramoth, 2003). In general, for the fungal control, it is always necessary to employ seeds with high physiological and sanitary quality, as well as crop rotation, resistant cultivars and 53

chemical treatment of seeds with fungicides. Seed treatment has a low cost and can improve seed germination and seedling development. However, for effective seed treatment and fungal control, effective fungicides are necessary (Athayde Sobrinho and Belmino, 201-?; Carvalho and Nakagawa, 2000; Menten and Moraes, 2010; Santos, 2009).

3.3.11 Occurrence regions and economic impacts: Brazil as case study In Brazil, Bipolaris sp. is more frequent in the Southern region, although with severity ranging from low to average. However, Exserohilum syn. Helminthosporium sp. can reduce up to 50% of the production, primarily in the second harvest of sorghum, in the Brazilian states of São Paulo, Pernambuco, and Distrito Federal, and of maize and tropical grasses in the Brazilian states of São Paulo and Minas Gerais, respectively (Cruz, 2007; Mendes and Urben, 201-?; Rosseto and Santiago, 201-?). At world level, Exserohilum syn. Helminthosporium sp. can also be found in the Dominican Republic (Barbosa, 2007). The fungus Curvularia sp. in tropical grasses can be found in the Brazilian states of Mato Grosso, Minas Gerais, and São Paulo, as well as in maize crops in the states of Mato Grosso, Mato Grosso do Sul and Pernambuco (Rosseto and Santiago, 201-?). The worldwide distribution of this fungus occurs in tropical and subtropical regions (Maia et al., 201-?). Phoma sp. occurs both in the European region and in Brazil (Aveskamp et al., 2008; Irinyi et al., 2009 Salgado and Pfenning, 2000; Vilela Júnior, 2011). Reported in Honduras, Costa Rica, Panama, and Colombia in the 1960s, Fusarium sp., has primarily reached the Northeast region of Brazil (Castro et al., 2008). The occurrence of Macrophomina sp. associated with soybean crops can be found in the Center-West region of Brazil, besides the states of Paraná, Rio Grande do Sul, São Paulo, Minas Gerais, and Maranhão. The association with cotton is reported in São Paulo, Paraíba, Pernambuco, Minas Gerais, and Paraná. The association with bean crops might occur in the entire Brazilian territory, except for the states of Amazonas, Roraima, Amapá, Rondônia, and Acre. The association with maize might occur in the South, Southeast and Central-West regions, besides the states of Pernambuco and Bahia. The association with sunflower is verified in Mato Grosso, São 54

Paulo, and Paraná. The association with sorghum is reported in the Brazilian Northeast region and in the state of Rio Grande do Sul (Mendes and Urben, 201-?). It can also be verified causing damage in Venezuelan crops (Barbosa et al., 2007). The presence of Rhizoctonia sp. associated with tropical grasses can be verified in the Brazilian states of Pará and Mato Grosso; in the soybean crop it presents a wide distribution throughout the Brazilian territory, except in the states of Goiás, Espírito Santo, and Rio de Janeiro; as for cotton crops, it only occurs in the state of Paraná (Mendes and Urben, 201-?). There are also incidence and damage reports of Rhizoctonia sp. and Pythium sp. in the state of Florida, in the United States of America (Barbosa et al., 2007). Furthermore, the fungus Rhizoctonia sp. might cause economic impacts through the reduction of the initial plant population, generating the need for resowing, which costs 6.44% of the total production cost (Goulart, 2016). The occurrence of Sclerotium sp. has increased in the last few years, and it has been isolated from several locations in the Mid-North region of Brazil (Athayde Sobrinho and Belmino, 201-?). Generally, the severe incidence of fungi might lead to the increase in the production costs through the need for resowing due to damage in the initial stand of the crops (Goulart, 2016); and through the need for the application of chemical (via seed and leaves) and biological treatments. Furthermore, it negatively affects the crop yield since it causes a reduction in the production of green mass and in the photosynthetic ability, with a reflection of these effects in the production and quality of the grains. The percentages of cost increase and yield reduction in the crops are variable and dependent on the level of incidence by the phytopathogens, interspecific interaction between pathogen and host, disease severity and abiotic conditions (Bettiol and Morandi, 2009; Casa et al., 2005; Costa, 2009; Reis et al., 2004; 2010; Uebel, 2015). Briefly, based on the scientific findings presented in this paper, the following table was generated (Table 1). 55

Table 1. Most occurring regions, susceptible crops, damages and control of the fungi discussed in this paper. Fungi Regions Susceptible Damages Controls crops Bipolaris sp. Regions with a Tropical grasses, In plants: brown Resistant cultivars; temperatures between 22 and maize, wheat, rice and spots and coalescence of the Fungicides tebuconazole; 30 °C; Brazil coffee lesions; In animals and pyraclostrobin with epoxyconazole humans: allergies, pulmonary and cutaneous infections Curvularia sp. Found in several regions Tropical grasses In plants: leaf spot Fungicides thiram; thiram of the world; tropical and and maize disease; In animals and with carboxin; fludioxonil with subtropical regions; Brazil humans: allergic conditions, metalaxyl-M and thiabendazole. urinary and pulmonary infections, among others Exserohilum sp. Regions with a Tropical grasses, In plants: leaf and Genetic resistance; crop temperatures between 18 and sorghum and maize stem spot diseases, rotting, rotation 27 °C and high moisture; Brazil seed unfeasibility and death and Dominican Republic of seedlings Phoma sp. Brazil and European Soybean and In plants: leaf spot, Fungicides thiram; thiram region coffee rotting, seed unfeasibility and with carboxin and fludioxonil with death of seedlings metalaxyl-M Fusarium sp. Regions with high Pine, cotton, In plants: red root rot Resistant cultivars; healthy temperature and soil moisture; wheat, rice, bean, and fusarium rot disease; In and certified seeds; Fungicides Honduras, Costa Rica, Panama, soybean, maize, animals: intoxication due thiram; carboxin with thiram and Colombia and Brazil sugarcane, sorghum, and mycotoxins fludioxonil with metalaxyl-M tropical grasses Macrophomina sp. Regions with high Sunflower, cotton, In plants: rot and Cultivars with higher temperatures and moisture; sorghum, maize, soybean tissue destruction of roots, tolerance to drought and/or high Brazil and Venezuela and bean stems, leaves, and fruits temperatures

Table 1. Most occurring regions, susceptible crops, damages and control of the fungi discussed in this paper. Pythium sp. Regions with high Soybean, In plants: chlorosis, Chemical treatment of seeds; moisture and mild temperature; sorghum, cotton, bean, growth reduction, wilt, root rotation of crops; elimination of crop Brazil and United States of maize, wheat and tropical rot and tipping residues; biological control with America grasses Trichoderma spp. Rhizoctonia sp. Brazil and United States Tropical grasses, In plants: leaf Chemical treatment of seeds; of America soybean, maize, and burning, rot, tipping, inhibit rotation of crops; elimination of crop cotton growth and death residues; biological control with Trichoderma spp. Sclerotium sp. Regions with Soybean, bean, In plants: cortical rot, Healthy and certified seeds; temperatures within 25 and 35 potato, and tomato wilt, drought and death elimination of crop residues; crop °C; Brazil rotation; fungicide tebuconazole 57

3.4 Conclusion The main fungi reported to be associated with forage and agricultural crops and soils of integrated systems in Brazilian tropical regions are Bipolaris sp., Curvularia sp., Exserohilum syn. Helminthosporium sp., Phoma sp., Fusarium sp., Macrophomina sp., Pythium sp., Rhizoctonia sp. and Sclerotium sp. The main methods of fungal control involve the employment of high-quality seeds, crop rotation, resistant cultivars and chemical seed treatment.

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4. EFFECT OF FERTILIZER ON PALISADEGRASS SEEDS PATHOGENS

ABSTRACT: This study aimed to evaluate the effects of the contact of Brachiaria seeds with the fertilizer 05-25-15 on the sanitary quality of the seeds. The experimental design was completely randomized, in a (5x2) factorial design, with four replications. The treatments consisted of five contact times (0, 24, 48, 72 and 96 hours) of the fertilizer with seeds of ruziziensis grass and marandu grass. After the contact times of the Brachiaria seeds with the fertilizer, the seeds from each species were separated and the following analyses were performed: water content, germination, seed health ("Blotter Test"), and accelerated aging, with later germination test and sanitary analysis of seeds. Data were analysed for variance and regression analysis at the significance level of 5% and descriptive analysis was used for the results of the sanitary analysis. The contact time reduces the incidence of pathogens in the seeds of ruziziensis grass and marandu grass treated with the fertilizer due to the salinity and acid pH of the fertilizer. The fungi related to the reduction in germination were Aspergillus sp. and Fusarium sp. The disinfestation process increased the incidence of Fusarium sp., under major internal infestation of this fungus in palisadegrass seeds.

Keywords: Accelerated aging, Aspergillus sp., Blotter Test, Brachiaria sp., Fusarium sp., germination.

EFEITO DO FERTILIZANTE NOS PATÓGENOS DAS SEMENTES DE CAPIM

RESUMO: Este trabalho objetivou avaliar os efeitos do contato das sementes de Brachiaria com o fertilizante 05-25-15 sobre a qualidade sanitária das sementes. O delineamento experimental foi inteiramente casualizado, em esquema fatorial (5x2), com quatro repetições. Os tratamentos consistiram de cinco tempos de contato (0, 24, 48, 72 e 96 horas) do fertilizante com sementes de capins ruziziensis e marandu. Após os tempos de contato das sementes de Brachiaria com o fertilizante, as sementes de cada espécie foram separadas e as seguintes análises foram realizadas: teor de água, germinação, sanidade das sementes ("Blotter Test") e envelhecimento acelerado, com posterior teste de germinação e sanidade. Os dados foram submetidos à análise de variância e regressão ao nível de significância de 5% e a análise descritiva foi utilizada para os resultados da análise sanitária. O tempo de contato reduz a incidência de patógenos nas sementes de capins ruziziensis e marandu tratados com o fertilizante, devido à salinidade e pH ácido do fertilizante. Os fungos relacionados à possível redução na germinação foram Aspergillus sp. e Fusarium sp. O processo de desinfestação aumentou a incidência de Fusarium sp., com maior infestação interna deste fungo em sementes de capim.

Palavras-chave: Aspergillus sp., Blotter Test, Brachiaria sp., envelhecimento acelerado, Fusarium sp., germinação.

4.1 Introduction The presence of degraded pastures is particularly common in agricultural frontier areas in the country, such as the North and Midwest regions. In these places, this phenomenon is directly associated with the low productivity of livestock and the increase in deforestation. Several sources indicate that, of the 172 million hectares of pastures in Brazil, more than 60% are in some stage of degradation. In Cerrado areas, which account for 60% of the country's meat production, around 80% of the 45-50 million hectares with cultivated pastures show some degree of degradation (Nogueira, 201-?), Integrated crop-livestock systems are a feasible alternative in order to promote socioeconomic and sustainable development before a growing food demand and the need to reduce deforestation (Vilela et al., 2011), increasing production system efficiency (Balbino et al., 2011). In addition to the financial benefits (Macedo, 2009; Vilela et al., 2011), there is synergism between pastures and annual crops, such as: improvement of physical, chemical and biological properties of soil, control of diseases, pests and weeds (Leonel, 2011). With the sustainable increase in Brazilian livestock production, comparing the data obtained in 2010 with the information from 2018, an improvement in the quality of pastures was observed in the evaluated period, with a reduction from 29.1% to 41.1% in the percentage of degraded areas (Ferreira Júnior, et. al, 2020). Based on the implantation premises of the Santa Fé system, where the grain crop is annually intercropped with a forage crop, stand out maize and Brachiaria (Syn. Urochloa) ruziziensis cv. Kennedy (Ruziziensis palisadegrass). For the deployment of the Barreirão system, in which a grain crop is intercropped and/or rotated with a forage crop that acts in the area as a perennial culture, the most used species are maize and Brachiaria (Syn. Urochloa) brizantha cv. Marandu (Marandu palisadegrass) (Freitas et al., 2005). In order to minimize problems arising from the forage sowing in integrated systems, such as the lack of uniformity in the initial stand due to the reduced size and low weight of the seeds, the simultaneous sowing of grain and forage seeds is commonly performed, associating them to the fertilizer applied at the grain crop sowing (Lima et al., 2000). 68

However, the contact of the seeds with the fertilizer can influence their sanitary quality, altering the incidence of pathogens associated to the seeds due to fertilizer salinity and pH. For the maize crop implantation in Mato Grosso state, Brazil, the fertilizer 05-25-15 (N-P2O5-K2O) is commonly used. Therefore, this study aimed to evaluate the effect of the contact time of fertilizer

05-25-15 (N-P2O5-K2O) on the sanitary quality of palisadegrass seeds.

4.2 Materials and Methods The experiment was conducted in a completely randomized design, with four replications. Treatments consisted of a 5x2 factorial design, with five contact times (0,

24, 48, 72 and 96 hours) of the fertilizer 05-25-15 (N-P2O5-K2O) on Brachiaria (Syn. Urochloa) ruziziensis cv. Kennedy (ruziziensis palisadegrass) and Brachiaria (Syn. Urochloa) brizantha cv. Marandu (Marandu palisadegrass) seeds. The fertilizer consisted of mono-ammonium phosphate, single superphosphate, triple superphosphate and potassium chloride, with a saline index of 70.59% and pH 4.83. The association of the seeds with the fertilizer was performed for both grass species, with the seeds being then transferred to sealed plastic packages and stored until reaching the pre-established contact times. Afterwards, the fertilizer was removed from the mixtures and the grass seeds were subjected to the tests of water content, germination, health ("Blotter Test" with salt stress) and accelerated aging analyses, with later germination test and analysis of sanitary conditions. In order to determine the water content, three 4.0 g samples were placed in a drying oven for 24 hours at a temperature of 105 ± 1 °C, for each species and treatment. After the drying process, the samples were placed in desiccators to promote the cooling and then weighed in analytical balance (0.0001 g). The results were expressed as percentage, according to the methodology of the authors cited in the reference (Brazil, 2009a). The methodology used for the germination test followed the described by the authors cited in the reference (Brazil, 2009a), in which four sub-samples of 50 seeds were used for each species and treatment. The seeds were placed equidistantly in a 69

polystyrene box (gerbox) on two sheets of blotter paper as substrate, and moistened with distilled water in the ratio of two and a half times the dry mass of the paper. Subsequently, the boxes were sealed with film paper to reduce moisture loss, and taken to the BOD (Biochemical Oxygen Demand) chamber, with photoperiod and temperature regulation (12 hours of light at 35 °C and 12 hours in the absence of light at 20 °C). Moisture inside the gerbox was maintained with the addition of distilled water. At 21 days the germinated seeds were evaluated, considering germinated the seeds that had emitted 2 mm of root. The results were expressed as percentage. Seed sanitary analysis was performed according to the methodology of the authors cited in the reference (Brazil, 2009b) modified for water restriction, according to the methodology of the authors cited in the reference (Machado et al., 2007). For each treatment and grass species, 100 pure and round seeds and 100 pure, round and disinfested seeds were used. The disinfestation process was performed in a laminar flow cabinet by immersing the seeds in a 1% sodium hypochlorite solution for three minutes. The seeds were then washed with sterile distilled water, according to the described by the authors cited in the reference (Brazil, 2009b). Afterwards, the seeds were distributed equidistantly in a gerbox containing three sheets of filter paper, previously moistened with a sterile sodium chloride solution (-0.6 MPa), according to the described by the authors cited in the reference (Machado et.al, 2007), in a proportion equivalent to two and a half times the dry mass of the substrate. During the analyses period, substrate moisture was maintained by the addition of a sterile sodium chloride solution (-0.6 MPa) to restrict seed germination and to ensure the accurate evaluation of microorganisms incidence. The seeds were incubated in a BOD chamber under the constant temperature of 20 °C and a 12-hour photoperiod. Seven days later, the individual evaluation of the seeds was performed with the aid of stereoscopic and biological microscopes. The fungi were identified by the observation of morphological structures, with the aid of specialized literature. Results were expressed as percentage of fungi incidence, according to the methodology of the authors cited in the reference (Brazil, 2009b). The accelerated aging test was performed according to the methodology proposed by the author cited in reference (Marcos Filho, 1999), in which the seeds of 70

each treatment were distributed on an aluminium screen attached to a gerbox containing 40 mL of distilled water. The boxes were then capped, forming a wet chamber, and placed in a BOD chamber for a period of 36 hours at a temperature of 42 °C. After the accelerated aging, germination and seed health tests, along with seed evaluation, were performed seven days later. Data were subjected to analysis of variance (ANOVA) and regression analysis at a significance level of 5% probability (P=0.05), and descriptive analysis for the results of the total sanitary analysis (high variability data).

4. 3 Results In the water content analysis, there was no effect of the contact time and no interaction between forages and contact times. A mean water content of 7.91% was observed. In the germination test there was an interaction effect of contact time x forage species. The contact time with the fertilizer did not affect the germination of the ruziziensis palisadegrass seeds, with mean value of 75.10%. However, the evaluation of the marandu palisadegrass seeds showed a reduction in germination percentage as the contact time of the seeds with the fertilizer was prolonged (Fig. 1). An interaction effect of contact time x forage species on the percentage of seed germination after the accelerated aging was observed. There was a quadratic effect in the contact time of ruziziensis palisadegrass seeds with the fertilizer on accelerated aging (Fig. 1), with a decrease in the germination percentage of aged seeds as the contact time with the fertilizer increased up to 42 hours, with minimum germination value of 66.50%. In the present study, the contact time with the fertilizer did not affect the germination of the marandu palisade grass seeds subjected to accelerated aging, with a mean germination value of 49.80%.

100 Ruziziensis palisadegrass Accelerated aging y= 0.0035x2* - 0.3208x* + 74.5 R² = 0.2409

40 Percentage

20 Marandu palisadegrass Seeds germination y= -0.275x*+ 56.8 R² = 0.4989 0 0 24 48 72 96 Time (hour)

Fig. 1. Germination of marandu palisadegrass seeds (A) and accelerated aging of ruziziensis palisadegrass seeds (B) as a function of the contact time with the fertilizer 05-25-15. *Significant at 5% probability (P = 0.05)

For the sanitary analysis of the seeds treated with fertilizer and for those treated with fertilizer and exposed to the accelerated aging, there was an interaction effect of the contact time x forage species on the percentage of fungi incidence, with the occurrence of the fungi Alternaria sp., Aspergillus sp., Bipolaris sp., Cladosporium sp., Cercospora sp., Fusarium sp., Nigrospora sp., Penicillium sp., Rhizoctonia sp. and Rhizopus sp., and predominance of fungi Aspergillus sp. and Fusarium sp. (Fig. 2), in both analyses and forage species. Fungal incidence data reflect the average incidence of fungi from the experimental treatments, presenting, therefore, high variability. Because of this, descriptive analysis was adopted. For Aspergillus sp., incidence percentages of 6.36 and 5.05% without disinfestation, and 2.96 and 2.05% with disinfestation were observed in the fertilizer- treated seeds of ruziziensis palisadegrass and marandu palisadegrass, respectively; and incidence percentages of 71.08% and 21.21% without disinfestation and 5.40% and 3.80% with disinfestation in the seeds treated with fertilizer and exposed to accelerated aging of ruziziensis palisadegrass and marandu palisadegrass, respectively. 72

Fig. 2. Fungi Aspergillus sp. (left and right) and Fusarium sp. (center) found in the grass seeds. There was a high percentage of Fusarium sp. in the seeds treated with fertilizer (78.66% and 81.33% in ruziziensis palisadegrass and marandu palisadegrass, respectively), and in the seeds treated with fertilizer exposed to accelerated aging (63.42% and 63.72%, in ruziziensis palisadegrass and marandu palisadegrass, respectively). The incidence of Fusarium sp. in the seeds remained high both in fertilized- disinfested seeds (73.33% and 62.33% in ruziziensis and marandu palisadegrass seeds, respectively), as in the seeds treated with fertilizer and exposed to accelerated and disinfested aging (35.20% and 40.23% in ruziziensis and marandu palisadegrass seeds, respectively). The increase in the contact time of the marandu and ruziziensis palisadegrass seeds with the fertilizer decreased the incidence of fungi (Fig. 3). When comparing the incidence of fungi in the absence of contact with the fertilizer (time zero) with the maximum studied time (96 h), a reduction of 18.67 and 27.38% was observed in the seeds of ruziziensis palisadegrass and marandu palisadegrass, respectively. With regard to the seeds of ruziziensis and marandu palisadegrass treated with fertilizer and exposed to accelerated aging, when comparing to the incidence of fungi in the absence of contact with the fertilizer (time zero) in the maximum studied time (96 h), an increase of 13.00% and a reduction of 5.42% were observed in the seeds of ruziziensis palisadegrass and marandu palisadegrass, respectively.

100 y Marandu= -0.1507x* + 80.872 R² = 0.7437

y Ruziziensis= -0.2863x* + 81.202 40

R² = 0.8074 Incidence Incidence (%)

0 0 24 48 72 96 Time (hour)

Fig. 3. Percentage of fungi incidence in ruziziensis and marandu palisadegrass seeds after the treatments as a function of the contact time with the fertilizer 05-25- 15. *Significant at 5% probability (P = 0.05).

100

60 y Ruziziensis= 0.0434x* + 32.07 R² = 0.4042

40 Incidence Incidence (%)

20 y Marandu= -0.0142x* + 23.822 0 R² = 0.0119 0 24 48 72 96 Time (hour)

Fig. 4. Percentage of fungi incidence in aged seeds of ruziziensis and marandu palisadegrass as a function of the contact time with the fertilizer 05-25-15. * Significant at 5% probability (P = 0.05). 74

When analysing the accelerated aging test, the germination percentage of the ruziziensis palisadegrass seeds was reduced with contact time up to 42 hours, possibly due to interference of the intense sporulation of external pathogens after the accelerated aging, confirmed by the sanitary analysis (Fig. 4).

4.4 Discussion Seed contact time with the fertilizer does not interfere in their water content, although the utilized fertilizer consists of single and triple superphosphate and potassium chloride, besides having a high saline index (70.59%) and acidic pH (4.83). Conversely, the authors cited in the reference (Dan et al., 2011), when studying ruziziensis palisadegrass seeds subjected to contact with urea, obtained positive linear effect for the water content as a consequence of the high hygroscopicity of the fertilizer. The authors cited in the references (Lima et al., 2009; 2010; 2013; Peres et al., 2012) observed an increase in the water content of marandu palisadegrass seeds during the contact time with fertilizers due to the high urea hygroscopicity, to the acid phosphate fertilizers obtainment, and to the high saline index of potassium chloride. In the evaluation of the germination percentage of ruziziensis palisadegrass seeds, it was verified that there was no effect of the seeds contact time with the fertilizer on germination. However, a reduction in germination was observed for the marandu palisadegrass seeds as the contact time with the fertilizer increased (Fig. 1), due to tegument rupture and extravasation of electrolytes by saline effect (Mateus et al., 2007). The authors cited in the references (Lima et al., 2009; 2010; 2013) verified a reduction in the germination percentage of marandu palisadegrass as the seeds contact time with the fertilizers was prolonged, corroborating with the present study. The same phenomenon was not observed for the ruziziensis palisadegrass seeds, probably due to the tegument being less susceptible to damage by intrinsic factors to the fertilizer, such as acid pH and saline effect. Further studies on tegument constitution of the species used in the present study are necessary. There was no effect of seeds contact time with the fertilizer on the germination of the marandu palisadegrass seeds subjected to accelerated aging. This phenomenon 75

can be related to the low sporulation of external pathogens, as presented in Fig. 4, derived from the sanitary quality of the seeds. Based on this, the importance of seed health can be endorsed, since the aging test predicts the behaviour of stored seeds; furthermore, an interaction between germination decrease of aged seeds and low sanitary quality of seeds was verified. From the seeds sanitary analysis, it was verified that the results of fungi incidence obtained by the authors cited in the reference (Dias et al., 1993; Mallmann et al., 2013; Santos et al., 2014; Sbalcheiro et al., 2014) corroborate with those found in the present study (Alternaria sp., Aspergillus sp., Bipolaris sp., Cladosporium sp., Cercospora sp., Fusarium sp., Nigrospora sp., Penicillium sp., Rhizoctonia sp. e Rhizopus sp.). The authors cited in the reference (Dias et al., 1993) have similarly identified the fungi Alternaria tenuis, Aspergillus sp., Cladosporium sp., Fusarium sp., Penicillium sp. and Rhizopus sp. in Brachiaria (Syn. Urochloa) brizantha seeds, whereas other authors, cited in the reference (Mallmann et al., 2013), reported a similar incidence of Bipolaris sp. and Cladosporium sp. in marandu palisadegrass seeds produced in Mato Grosso, besides Alternaria sp. and Fusarium sp. in seeds of Brachiaria (Syn. Urochloa) sp. and Panicum syn. Mehathyrsus maximum. In studies performed by the authors cited in the reference (Santos et al., 2014; Sbalcheiro et al., 2014), they verified the presence of the fungi Aspergillus niger, Bipolaris sp., Fusarium sp., Penicillium sp. and Rhizopus sp. in Brachiaria (Syn. Urochloa) brizantha cv. BRS Piatã (piatã palisadegrass). In addition, the authors cited in the reference (Sbalcheiro et al., 2014) observed the presence of Alternaria sp., Aspergillus flavus, Aspergillus ochraceus and Cladosporium sp. The main fungi evidenced in this work were Aspergillus sp. and Fusarium sp. in both forage species, and can be related to the reduction in seed germination (Sbalcheiro et al., 2014), causing damage to the quality and establishment of forage crops (Marchi et al., 2010; Santos et al., 2014). In addition to the physiological damage caused to Brachiaria seeds, both fungi can produce mycotoxins under low humidity conditions, which can lead to intoxication, cancer and death if ingested by animals (Vechiato and Aparecido, 2010). 76

The fungi that can be transmitted by seeds, with infestation/infection capacity during storage and in the physiological maturity point, may interfere in seed quality, reducing germination and forage production by compromising plant establishment, especially under favourable development conditions and with inefficient control methods (Marchi et al., 2007; 2010; Sbalcheiro et al., 2014). In the transmission of seed pathogens to seedlings, the authors cited in the reference (Sbalcheiro et al., 2014) reported the occurrence of pathogens Aspergillus sp. and Fusarium sp. It is observed that the use of low sanitary quality seed results in unsuccessful pasture formation and seed lots commercialization, due to the presence of fungi (Mallmann et al., 2013). Based on the seeds treated with fertilizer and subjected to accelerated aging, a high incidence of Aspergillus sp. on the surface of the Brachiaria seeds may occur due to the high incidence of this pathogen after the processes of disinfestation and accelerated aging on the seeds (ruziziensis palisadegrass presented 71.08 and 5.40 %, before and after disinfestation, respectively; marandu palisadegrass presented 21.01 and 3.80 %, before and after disinfestation, respectively). In the accelerated aging, seeds are subjected to high temperature and humidity, which are favourable conditions for sporulation and development of pathogens, especially in the outer layer of seeds. The contamination of the seeds by Aspergillus sp. can cause damage to the their physiological quality, reducing germination and vigour (Vechiato and Aparecido, 2010), causing a reduction of the planting stand, as well as being an inoculum source for the development of diseases and introducing pathogens in unaffected regions (Soave and Whetzel, 1987), such as the rot disease in corn ears (Costa et al., 2009). The incidence of Fusarium sp. in Brachiaria seeds (treated with fertilizer and treated with fertilizer and exposed to the accelerated aging) remained high after the disinfestation process (ruziziensis palisadegrass presented 78.66 and 73.33 %, before and after disinfestation, respectively; marandu palisadegrass presented 81.33 and 62.33 %, before and after disinfestation, respectively), evidencing the possibility of major incidence within the interior of the seeds. As a consequence of fungi incidence there may be a reduction in the viability percentage of the lots or death of the Brachiaria 77

seeds, once Fusarium sp. is a soil fungus that can be associated with the seeds (Costa et al., 2009; Verzignassi and Fernandes, 2001). Since it presents alternative hosts (maize, sorghum, sugarcane, grass, among others), crop rotation is not a very efficient control practice in these cases. Among them, Fusarium moniliforme and Fusarium graminearum stand out for causing stalk and root rot to infected plants (Costa et al., 2009), and Fusarium clamydosporium for causing wilt disease symptoms followed by death in forage plants such as Stylosanthes sp. (Verzignassi and Fernandes, 2001). The effect of seed dormancy breaking due to the accelerated aging process was excluded, since the germination percentage of ruziziensis palisadegrass seeds subjected to accelerated aging in the absence of contact (time zero) with the fertilizer was lower (74.90%) than the germination percentage obtained in the germination test (75.10%) (Fig. 1). The authors cited in the reference (Meschede et al., 2004) observed a reduction in the germination percentage of marandu palisadegrass seeds after 24 hours of aging at 43 °C. It is observed that the contact of the marandu and ruziziensis palisadegrass seeds with the fertilizer improved seed sanitization, since the prolongation of the contact time with the fertilizer decreased the incidence of fungi (Fig. 3). The increase in the contact time of the Brachiaria seeds exposed to accelerated aging leads to an increase in the fungi incidence on ruziziensis palisadegrass seeds and reduces the fungi incidence on marandu palisadegrass seeds (Fig. 4). This effect can be attributed to the accelerated aging process of the seeds treated with fertilizer, which promotes proliferation and development of fungi due to the optimal conditions provided (Fig. 4).

4.5 Conclusion The presence of the fungi Bipolaris sp., Fusarium sp., Rhizoctonia sp., Cercospora sp., Alternaria sp., Aspergillus sp., Cladosporium sp., Penicillium sp., Rhizopus sp., and Nigrospora sp was verified in the seeds of the ruziziensis and marandu palisadegrasses treated with fertilizer, as well as in the seeds treated with the fertilizer and exposed to accelerated aging. 78

The fungi related to the reduction in the germination of Brachiaria seeds are Aspergillus sp. and Fusarium sp., with a major internal infestation of Fusarium sp. It was verified that the increase in the contact time of the Brachiaria seeds treated with fertilizer reduces the incidence of fungi, improving the sanitary quality of the seeds.

4.6 References Balbino LC, Barcellos AO, Stone, LF. Reference frame: crop-livestock-forest integration. Brasília: Embrapa; 2011. English.

Brazil Ministry of Agriculture, Livestock and Food Supply. Rules for seed analysis. Brasilia DF. MAP / ACS. 2009a. Portuguese.

Brazil Ministry of Agriculture, Livestock and Food Supply. Rules for sanitary seed analysis. Brasilia DF. MAP / ACS. 2009b. Portuguese.

Costa RV, Casela CR, Cota LV. Corn Crops: Diseases. In: Cruz JP, Magalhães PC, Pereira Filho IA, Moreira JAA, editors. Sete Lagoas, Embrapa. 2009;138-169.

Dias DCFS, Toledo FF. Germination and incidence of fungi in tests with Brachiaria brizantha (Hochst. ex A. Rich) seeds Stapf. Scientia Agrícola. 1993;1(3):68-76. DOI: 10.1590 / S0101-31222008000300019.

Freitas FCL, Ferreira LR, Ferreira FA, Santos MV, Agnes EL, Cardoso AAet al. Pasture formation via Brachiaria brizantha consortium with corn for silage in no - tillage system. Planta Daninha. 2005;23:(1)49-58. Portuguese. DOI: 0.1590 / S0100- 83582005000100007.

Ferreira Júnior, LG, Oliveira-Santos C, Mesquita VV, Parente L.L. Dynamics of Brazilian pastures: Occupation of areas and signs of degradation - 2010 a 2018. Lapig: UFG, 2020. 19p. Portuguese.

Leonel FP. Integration of crop-livestock: production and quality of fodder. In: Zervoudakis JT, Cabral LS, editors. Nutrition and production of bovine cuttings. Cuiabá: 2011;221-233.Portuguese.

Lima EDV, Tavares JC, Azevedo VR, Leitão-Lima PS. Mixture of Brachiaria brizantha seeds with NPK fertilizer. Rural Science. 2010;40(2):471-474. English. DOI: 10.1590 / S0103-84782010005000003.

Macedo MCM. Crop-livestock integration: the state of the art and technological innovations. Revista Brasileira de Zootecnia. 2009;38:133-146. Portuguese. DOI: 10.1590 / S1516-35982009001300015.

Machado AQ, Machado JC, Vieira MDGGC, Cassetari Neto D, Souza MV. Potential of the use of water restriction in sanitary tests of cotton seeds. Plant Phytopathology. 2007; 32(5):408-414. Portuguese. DOI: 10.1590 / S0100-41582007000500006.

Marchi CE, Fernandes CD, Borges CT, Santos JM, Jerba VF, Trentin RA, Guimarães, L.R.A. Nematofauna pathogenic of tropical forage commercial seeds. Pesquisa Agropecuária Brasileira. 2007;42:655-660.Portuguese. DOI: 10.1590 / S0100- 204X2007000500007.

Marchi CE, Fernandes CD, Bueno ML, Batista MV, Fabris LR. Fungi propagated by commercial seeds of Brachiaria. Arquivo Instituto Biológico. 2010;77:(1)65-73. Portuguese.

Marcos Filho J. Accelerated aging test. In: Krzyzanowski FC, Vieira RD, France Neto JB. Seed vigor: concepts and tests. Londrina: Abrates; 1999. Portuguese.

Mateus GP, Borghi E, Marques RR, Bôas RLV, Crusciol CAC. Fertilizer sources and contact periods and seed germination of Brachiaria brizantha. Brazilian Journal of Soil Science. 2007;31(1):177-183. English. DOI: 10.1590 / S0100-06832007000100018.

Meschede DK, Sales JGC, Braccini ADL, Scapim CA, Schuab SRP. Treatments to overcome the dormancy of the seeds of Brachiaria grass marandu cultivar. Brazilian Journal of Seeds. 2004;26(2):76-81. Portuguese.

Nogueira SF. Extensive cattle ranching and the panorama of pasture degradation in Brazil. Embrapa Monitoramento por Satélite 201- ?. Available in: 80

http://www.diadecampo.com.br/zpublisher/materias/Materia.asp?id=28010&secao=Artig os%20Especiais#:~:text=A%20presen%C3%A7a%20de%20pastagens%20degradadas, regi%C3%B5es%20Norte%20e%20Centro%20Oeste.&text=Diversas%20fontes%20indi cam%20que%2C%20dos,em%20algum%20est%C3%A1gio%20de%20degrada%C3% A7%C3%A3o. Accessed April 20, 2021. Portuguese.

Soave J, Whetzel MMVS. Pathology of seeds. Campinas: Cargill Foundation. 1987. Portuguese.

Vechiato MH, Aparecido CC, Fernandes CD. Frequency of fungi in commercial seed lots of Brachiaria and Panicum. Campo Grande: Embrapa. 2010. Portuguese.

Verzignassi JR, Fernandes CD. Diseases in forages. Campo Grande, Embrapa. 2001.

Vilela L, Martha Junior GB, Macedo MCM, Marchão R L, GuimarãesJúnior R., Pulrolnik, K et al. Integration systems for livestock farming in the Cerrado region. Agricultural Research. 2011;46(10):1127-1138. English. DOI: 10.1590 / S0100- 204X2011001000003.

5. IMPACT OF PHYTOPATHOGENS ON THE SEED QUALITY OF TROPICAL GRASSES IN INTEGRATED SYSTEMS

ABSTRACT: This work aimed to identify the main fungi and phytonematode genotypes associated with the main tropical forage seeds used in integrated systems, as well as to distinguish the seeds with best physiological and sanitary quality. Two experiments were conducted in a completely randomized design. Since, experiment 1 corresponds to the seeds from the 2017/18 crop and experiment 2 to the seeds from the 2018/19 crop. Each experiment was performed with four treatments and four replications per experiment. The treatments consisted of seeds of Brachiaria ruziziensis and Brachiaria brizantha, cultivars Marandu, Xaraés and BRS Piatã (Ruziziensis, Marandu, Xaraés, and Piatã grasses). The seeds were purchased in commercial packaging, belonging to the 2017/18 and 2018/19 crop years, from the same productive field. For each experiment, the following variables were evaluated: water content, cultural value based on germination, cultural value based on viability, physical purity, germination rate index (GRI), first count and percentage of germination, viability (tetrazolium), dormancy, accelerated aging, fungal incidence, and incidence of phytonematodes. Cladosporium sp., Fusarium sp., Rhizoctonia sp., Meloidogyne sp., Filenchus sp., Aphelenchus sp., Aphelencoides sp., and Rotylenchulus sp. are the main phytopathogens associated with the seeds studied in this work.Comparing the seeds studied in this work, the seeds of ruziziensis grasses have better physiological quality and the seeds of xaraés grasses have better sanitary quality.

Keywords: Fungal plant pathogens, germination, tropical pastures

IMPACTO DE FITOPATÓGENOS NA QUALIDADE DE SEMENTES DE CAPINS TROPICAIS EM SISTEMAS INTEGRADOS

RESUMO: Objetivou-se identificar os principais gêneros de fungos e fitonematoides associados às principais sementes forrageiras tropicais utilizadas em sistemas integrados, e distinguir as sementes com melhor qualidade fisiológica e sanitária. Foram realizados dois experimentos em laboratório com delineamento de modo inteiramente casualizado. Sendo que, o experimento 1 corresponde as sementes oriundas da safra 2017/18 e o experimento 2 às sementes da safra 2018/19. Cada experimento foi realizado com quatro tratamentos e quatro repetições em cada experimento. Os tratamentos consistiram nas sementes de Brachiaria ruziziensis e Brachiaria brizantha cultivares Marandu, Xaraés e BRS Piatã (capins Ruziziensis, Marandu, Xaraés e Piatã). As sementes foram adquiridas em embalagem comercial, oriundas das safras 2017/18 e 2018/19, do mesmo campo produtivo. Em cada experimento foram mensuradas as variáveis: teor de água, valor cultural baseado na germinação, valor cultural basead na viabilidade, pureza física, índice de velocidade de germinação, “primeira contagem” e porcentagem de germinação, viabilidade (tetrazólio), dormência, envelhecimento 82

acelerado, incidência fúngica e de fitonematoides. Cladosporium sp., Fusarium sp., Rhizoctonia sp., Meloidogyne sp., Filenchus sp., Aphelenchus sp., Aphelencoides sp. e Rotylenchulus sp. são os principais fitopatógenos encontrados em associação às sementes estudadas neste trabalho. Comparando as sementes estudadas neste trabalho, as sementes de capim-ruziziensis possuem melhor qualidade fisiológica e as sementes de capim-xaraés possuem melhor qualidade sanitária.

Palavras-chave: capins tropicais, germinação, patógenos fúngicos de plantas.

5.1 Introduction The presence of degraded pastures is particularly common in agricultural frontier areas in the country, such as the North and Midwest regions. In these places, this phenomenon is directly associated with the low productivity of livestock and the increase in deforestation. Several sources indicate that, of the 172 million hectares of pastures in Brazil, more than 60% are in some stage of degradation. In Cerrado areas, which account for 60% of the country's meat production, around 80% of the 45-50 million hectares with cultivated pastures show some degree of degradation (Nogueira, 201-?). Over the years, there has been a need to maximize the food outcome in response to an intensified livestock production, while ruling out the need to open new areas to meet this demand. In this regard, sustainable crop models such as the crop-livestock integration (CLI) have been developed, allowing the recovery of degraded areas, the intensification of pasture production and, consequently, of animal protein (Macedo, 2009; Balbino et al., 2011; Leonel, 2011; Vilela et al., 2011). With the sustainable increase in Brazilian livestock production, comparing the data obtained in 2010 with the information from 2018, an improvement in the quality of pastures was observed in the evaluated period, with a reduction from 29.1% to 41.1% in the percentage of degraded areas (Ferreira Júnior, et. al, 2020). Although economically expressive, the system of forage seeds production is threatened by the presence of pathogens. Due to the absence of sanitary standards in Brazilian trade regulations, the seeds have acted as an efficient mechanism of introduction and dispersion of pathogens that can affect several moments, ranging from the stage of stand establishment to the harvest of the product (Vechiato and Aparecido, 2008). 83

Among the pathogens associated with tropical forage seeds of the genus Brachiaria syn. Urochloa stand out fungi and phytonematodes. When present in the seeds, the fungi can cause a reduction of their germination, diminishing its quality and commercial value (Lasca et al., 2004). In addition, it is believed that phytonematodes can have a significant impact on dry matter production and on the persistence of the forage in the field (Favoreto, 2004). In order to establish the appropriate strategies to eradicate fungi and phytonematodes from the production field, it is important to analyze the sanitary quality and the inference on the physiological quality of the seeds of the main tropical forages cultivated in integrated systems. Based on this, this work aimed to identify the main fungi and phytonematodes genera associated with the main tropical forage seeds employed in integrated systems, as well as to distinguish the seeds with best physiological and sanitary quality.

5.2 Material and Methods Two experiments were conducted in a completely randomized design. Each experiment was performed with four treatments and four replications per experiment. The treatments consisted of the seeds of Brachiaria ruziziensis (Ruziziensis grass) and Brachiaria brizantha, cultivars Marandu, Xaraés, and BRS Piatã (Marandu, Xaraés, and Piatã grasses), which are commonly used in integrated crop-livestock systems in tropical regions. The seeds were purchased in commercial packaging, belonging to the 2017/18 (Year 1: Experiment 1) and 2018/19 crop years (Year 2: Experiment 2), with the same origin (production field) (Mato Grosso, Brazil). The following variables were evaluated in each experiment: water content, cultural value based on germination, cultural value based on viability, physical purity, germination rate index (GRI), first count and percentage of germination, viability (tetrazolium), dormancy, accelerated aging, fungal incidence, and incidence of phytonematodes. The determination of the water content occurred out of three 4.0 g samples per treatment, placed in an oven for 24 hours at a temperature of 105 ± 1 °C. Afterward, the 84

weighing was performed in an analytical balance (0.0001 g). The results were expressed as a percentage (Brasil, 2009b). The cultural value (CV) of the seeds was determined by purity, germination and viability tests. The purity test was performed by means of two sub-samples of 5.0 g per treatment, manually separating the impurities, other seeds and pure seeds. The portion of pure seeds was expressed as a percentage (Brasil, 2009b). The germination test was performed with four sub-samples of 50 seeds for each treatment. The seeds were equidistantly placed in gerbox boxes over two sheets of blotting paper, moistened with distilled water in the ratio of two and a half times the dry mass of the paper. The gerbox boxes were then placed in a BOD (Biochemical Oxygen Demand) chamber with a 12-hour photoperiod and alternating temperature of 35/20 °C. Germinated seed counts were daily performed in order to calculate the germination rate (Maguire, 1962). The first germination count (vigor index) was performed at seven days, whereas the last count was performed at the end of 21 days in order to determine the percentage of germination. The seeds were considered germinated after the emission of 2 mm of the primary root. The results were expressed as a percentage (Brasil, 2009b). The viability of the seeds was determined by the tetrazolium test with the non- germinated seeds at the end of the germination test. A longitudinal cut was made on the seeds and only one of its parts was placed in contact with the solution of tetrazolium salt at 0.5 % (2, 3, 5 triphenyl tetrazolium chloride). The seeds emerged in tetrazolium salt were conditioned in a BOD chamber for 3 hours, without light, at a temperature of 30 °C. At the end of the period, they were classified as viable or non-viable seeds. The results were expressed as a percentage (Brasil, 2009b). Seed dormancy was determined as the difference between the percentage of viable seeds and the percentage of germinated seeds. In the accelerated aging test, the seeds of each treatment were distributed on an aluminum screen coupled to gerbox boxes, with 40 mL of distilled water at the bottom of the container. The boxes were then covered and placed in a BOD chamber for 36 hours at 42 °C (Marcos Filho, 1999). Finally, the germination test was performed through the counting of the germinated seeds at seven days (Brasil, 2009b). 85

The incidence of fungi was analyzed using an adapted “blotter test”, with and without previous disinfestation. Disinfestation was performed by immersing the seeds in a 1 % sodium hypochlorite solution for 3 minutes (Brasil, 2009a). Four sub-samples of 25 seeds were used for each treatment. The seeds were equidistantly placed in gerbox boxes on two sheets of blotting paper previously moistened with a previously sterilized sodium chloride (NaCl) solution in the potential of - 0.6 MPa (water restriction) (Adapted from Machado et al., 2007), in the proportion of two and a half times the dry mass of the paper. The gerbox boxes were then placed in a BOD chamber with a 12-hour photoperiod and at a temperature of 20 °C (adapted from Brasil, 2009a). After seven days, the individual evaluation/examination of the seeds was performed with the aid of a magnifying glass, due to the occurrence of typical fruiting bodies of fungi growth, with these being grouped according to the genus. The observation of slides on a light microscope was necessary in order to confirm the identity of the fungi. The results were expressed as the percentage of occurrence (Brasil, 2009a). The incidence of phytonematodes was analyzed by the extraction and wet sieving of the samples, with ten replicates of 10 g of commercial seeds per treatment (Coolen and D‟Herde, 1972), with and without an incubation period. To every 10 g sample of the commercial seeds, one liter of water was added. Afterward, the solution was sifted on sieves of 20 and 500 mesh sizes. From the suspension retained in the 500-mesh sieve, an aliquot of 1 mL was withdrawn, and the identification and quantification of the phytonematodes was performed on a light microscope, down to the genus rank. The sample retained in the 20-mesh sieve was collected and incubated in 50 mL of water for 24 hours at a temperature of 25 °C. After the incubation period, the samples were immersed in approximately 200 mL of water. The solution was then sieved in 20, 60 and 400 mesh size sieves, and the suspension of the phytonematodes was retained in the 400-mesh screen. This suspension was centrifuged at 1,800 rotations per minute (rpm) for 5 minutes. Afterward, 86

the supernatant was discarded and a 45 % sucrose solution was added to the suspension. The mixture was homogenized and centrifuged at 1,800 rpm for 1 minute. The supernatant obtained after the procedure was poured into a 400-mesh sieve, and the retained phytonematode suspension was collected. An aliquot of 1 mL from this suspension was placed on a microscope slide for identification and quantification of the phytonematodes under an optical microscope, down to the genus rank. The data obtained on all variables, in both experiments, were transformed using the formula √x+0.5 and subjected to analysis of variance and Tukey‟s range test at the 5 % probability level. SISVAR computer software was utilized for the analyses.

5.3 Results In experiment 1, using the seeds of the 2017/2018 crop year, there was an effect of the treatment over the following variables: water content, purity, first germination count, germination and germination rate index, viability, dormancy, cultural value based on germination, cultural value based on viability, and germination of aged seeds of Brachiaria (p<0.05) (Table 1). In experiment 2, using the seeds of the 2018/2019 harvest, there was also a treatment effect over all variables, except for the water content and the cultural value based on viability (p<0.05) (Table 1). The Ruziziensis grass had the lowest water content (10.21%) among the studied species/cultivars, in experiment 1. In experiment 2, the mean value of the water content was 9.58 %. In experiment 1, it was observed that none of the lots reached the minimum purity index of 80% required for the commercialization of the seeds of Brachiaria sp. (Brasil 2008). In addition, considering that in order to commercialize the seeds of Brachiaria sp. the minimum standard of germination or viability is 60 % (Brasil, 2008; Brasil, 2010), it was verified that the Xaraés grass lot was below the germination standard, whereas the Marandu grass lot was not within the germination and viability standards for commercialization (Table 1).

TABLE 1. Water content (WC), purity analysis (P), viability (V), first count of germination (1stG), germination (G), germination rate index (GRI), cultural value based on germination (CV1), cultural value based on viability (CV2), dormancy (D) and germination of aged seeds (GA) of seeds of Brachiaria syn. Urochloa brizantha cultivars Marandu, BRS Piatã and Xaraés; and Brachiaria syn. Urochloa ruziziensis cultivar Kennedy, originating from the 2017/2018 and 2018/2019 harvests. Express in percentage (Mato Grosso, Brazil) Species/cultivar WC P V 1stG G GRI CV1 CV2 D GA Experiment 1 – Harvests 2017/2018 Xaraés grass 10.60 a 66.06 b 64.00 a 28.50 b 49.00 c 35.94 b 32.37 ab 42.28 a 15.00 a 40.50 b Ruziziensis grass 10.21 b 69.43 b 76.00 a 72.00 a 75.00 a 75.05 a 52.07 a 52.77 a 1.00 b 76.00 a Piatã grass 10.75 a 62.97 b 75.50 a 64.00 a 73.50 b 75.37 b 46.28 ab 47.54 a 2.00 b 67.50 a Marandu grass 10.68 a 75.73 a 33.00 b 25.50 b 27.50 d 27.25 b 20.82 b 24.99 b 5.50 ab 31.50 b Experiment 2 – Harvests 2018/2019 Xaraés grass 9.03 97.35 a 57.50 b 2.50 c 24.50 b 19.79 c 23.85 b 55.98 33.00 a 7.50 c Ruziziensis grass 9.85 93.06 b 65.00 a 47.00 a 56.50 a 81.78 a 52.58 a 60.49 8.50 b 46.50 a Piatã grass 9.54 98.14 a 62.00 a 25.50 b 51.50 a 54.52 b 50.54 a 60.85 10.50 b 37.00 ab Marandu grass 9.91 92.87 b 61.50 b 3.00 c 20.5 b 14.76 c 19.03 b 57.12 41.00 a 5.50 bc Means followed by the same letter, lowercase in the column, do not differ statistically by the Tukey test (p<0.05). 88

Conversely, in experiment 2, all lots reached the minimum purity index (80%) required for seed commercialization. However, no lots reached the minimum germination index (60%), and the Xaraés grass lot was below the viability standards (60%) for commercialization (Table 1). The Marandu grass seeds had lower viability percentage results in experiment 1 (Table 1), whereas in experiment 2, besides the Marandu grass seeds, the Xaraés grass seeds also showed lower results of viability percentage (Table 1). This suggests that the Marandu and Xaraés grasses present high levels of non-viable (dead) seeds in the commercialized lots. It was also verified that the seeds of the Ruziziensis and Piatã grasses presented higher results of the first germination count, which is indicative of greater vigor. As to the experiment 2, the Ruziziensis grass presented a best percentage of germination in the first count. The percentage of germination was higher in the Ruziziensis grass seeds, in experiment 1. In experiment 2, the percentage of germination was higher in the seeds of the Ruziziensis grass along with the Piatã grass. When analyzing the germination rate index (GRI) of the two experiments, it is observed that the Ruziziensis grass has a higher GRI in both (Table 1). Purity, germination and viability tests were performed in order to determine the cultural value (CV) of the lots of Brachiaria seeds (Table 1). Germination is commonly used to determine the cultural value (CV = (purity x germination) / 100). However, the viability test, being the fastest and most practical analysis method, is the most used for the commercialization of forage seeds after the publication of the Normative Instruction Nº 30 (Brasil, 2008; Brasil, 2010). According to the calculation of the cultural value obtained by both methods (based on germination and viability), in experiment 1, it is verified that the seed lot of the Ruziziensis grass reached the minimum standard for commercialization of 48% (Brasil, 2008; Brasil, 2010). When analyzing the experiment 2, it was observed that when calculating the cultural value using germination as a base, only the lots of the Ruziziensis and Piatã grasses reached a commercialization minimum (48%) (Table 1). In addition, it was 89

observed that the Marandu grass presents the lowest cultural value for both methods and experiments (Table 1). In both experiments, the Xaraés and Marandu grasses had a higher frequency of dormancy (Table 1). Furthermore, a lower percentage of germination was observed after the accelerated aging of the Xaraés and Marandu seeds, in both experiments (Table 1). The sanitary analysis of the Brachiaria seeds without disinfestation, in experiment 1, verified the incidence of the fungi Alternaria sp., Aspergillus sp., Cladosporium sp., Colletotrichum sp., Curvularia sp. Fusarium sp., Penicillium sp., Rhizoctonia sp., and Rhizopus sp. The same fungi were found in experiment 2, except Alternaria sp. and Curvularia sp. When the previous disinfestation process was performed, in experiment 1, a lower incidence of all the above-mentioned fungi was verified, except for Cladosporium sp. and Rhizoctonia sp., which presented an increase in frequency, and Colletotrichum sp., Curvularia sp. and Penicillium sp., which were not detected. However, in experiment 2, a lower incidence of all fungi was verified, as well as the absence of the fungus Colletotrichum sp. In both experiments, with and without the disinfestation process, the fungi Cladosporium sp., Rhizoctonia sp., and Fusarium sp. predominated (Table 2). There was no treatment effect on the incidence of the fungus Rhizoctonia sp. There were high percentages of Fusarium sp. in the seeds of all analyzed species/cultivars, both with and without the disinfestation method, and in both experiments. The exception was the Xaraés cultivar, which presented a reduction of the fungal genus Fusarium sp., going through the disinfection process in both experiments (Table 2). There was no treatment effect on the incidence of Fusarium sp. in non-disinfested seeds. However, the Ruziziensis grass seeds showed a higher incidence of Fusarium sp. after the disinfestation process in both experiments (Table 2). 90

TABLE 2. Incidence (%) of fungi Cladosporium sp., Fusarium sp. and Rizoctonia sp. in seeds of Brachiaria syn. Urochloa ruziziensis; Brachiaria syn. Urochloa brizantha cv. Marandu, cv. Xaraés and cv. BRS Piatã, without and with disinfestation, originating from the 2017/2018 and 2018/2019 harvests (Mato Grosso, Brazil). Cladosporium sp. Fusarium sp. Rizoctonia sp. Species/cultivar Without † With ‡ Without † With ‡ Without † With ‡ Experiment 1 – Harvests 2017/2018 Xaraés grass 2.00 B 44.00 67.00 a 9.00 bB 18.00 a 0.00 b Ruziziensis grass 0.00 aB 5.00 b 62.00 60.00 A 3.00 23.00 Piatã grass 32.00 A 36.00 88.00 a 53.00 bB 1.00 26.00 Marandu grass 6.00 B 14.00 76.00 a 32.00 bB 22.00 b 25.00 a Experiment 2 – Harvests 2018/2019 Xaraés grass 36.00 b 2.00 Ca 74.00 a 0.00 Bb 22.00 b 3.00 a Ruziziensis grass 53.00 b 28.00 Aa 77.00 a 22.00 Ab 66.00 b 11.00 a Piatã grass 49.00 b 16.00 ABa 63.00 33.00 A 41.00 15.00 Marandu grass 50.00 b 5.00 BCa 87.00 a 28.00 Ab 44.00 b 10.00 a † Without: without disinfestation; ‡ With: with disinfestation; Means followed by the same letter, uppercase in the column and lowercase in row, within each fungi genus, do not differ statistically by the Tukey test (p<0.05).

With regard to the genus Cladosporium sp., in experiment 1, the non-disinfested seeds of the Piatã grass presented a high incidence rate. However, considering the seeds after the disinfestation process, there was no effect of the treatment on the incidence of Cladosporium sp. In experiment 2, there was no treatment effect on the incidence of Cladosporium sp. in non-disinfested seeds. Right after the disinfestation process, the seeds of the Piatã and Ruziziensis grasses showed a higher incidence of this phytopathogen (Table 2). After the disinfestation process in experiment 1, it is estimated that with the reduction in the infestation by Fusarium sp., the development of the fungi Cladosporium sp. and Rhizoctonia sp may have been propitiated. Conversely, in experiment 2, a reduction trend of the incidence of all major fungi was observed (Table 2). In the phytonematode analysis of experiment 1, the presence of eggs and free- living phytonematodes (non-parasites) was observed in all analyzed seed lots through the method without incubation period. There was no treatment effect on egg incidence, and there was a high incidence of free-living phytonematodes in the Ruziziensis grass seeds (Table 3). In experiment 2, with no incubation period, it was possible to verify the presence of eggs, free-living phytonematodes (non-parasites) and the phytonematode Aphelencus sp. in all seed lots. There was no treatment effect on egg incidence, but there was a high incidence of free-living phytonematodes and Aphelencus sp. in the seeds of the Ruziziensis and Piatã grasses (Table 3). After the incubation period, in experiment 1, the incidence of eggs and free-living phytonematodes was verified, with incidence predominance in the seeds of the Ruziziensis grass. In addition, the incidence of the phytopathogenic fungi Meloidogyne sp., Aphelenchus sp., and Filenchus sp. was observed in the seeds except for those of the Xaraés grass. However, there was no treatment effect on the incidence of phytopathogenic fungi for the studied species/cultivars (Table 3). In experiment 2, after the incubation period, the incidence of eggs and free-living phytonematodes was observed. There was no treatment effect on the incidence of eggs, and it was verified that the Xaraés grass presents a low incidence of free-living phytonematodes. 92

TABLE 3. Egg incidence, free-living nematodes (non-phytopathogenic) and phytopathogenic nematode Aphelencus sp. in seeds of Brachiaria syn. Urochloa ruziziensis; Brachiaria syn. Urochloa brizantha cv. Marandu, cv. Xaraés and cv. BRS Piatã, with and no incubation, originating from harvest 2017/2018 and 2018/2019. Express in unit g-1 of pure seeds (Mato Grosso, Brazil). Experiment Experiment 1 – Harvest 2017/2018 Experiment 1 – Harvest 2018/2019 No incubation With incubation No incubation With incubation Species/cultivar Eggs ns Free-living Eggs Free-living Eggs ns Free-living Aphelencus Eggs ns Free-living Xaraés grass 148 68 b 100 b 132 c 0.00 1.20 c 0.00 b 2.80 10.80 b Ruziziensis grass 156 468 a 396 a 489.60 a 0.40 17.40 a 0.40 a 6.00 184.00 a Piatã grass 108 16 b 108 b 208 c 0.80 34.40 a 0.00 ab 2.40 147.70 a Marandu grass 188 136 b 516 a 238.80 b 0.40 24.40 b 0.40 b 4.40 56.60 a Means followed by the same letter, lowercase in the column, do not differ statistically by the Tukey test (p<0.05); ns: not significant (p<0.05).

Furthermore, the incidence of the phytopathogenic fungi Aphelenchus sp., Aphelencoides sp., Filenchus sp., and Rotylenchulus sp was also verified. However, there was no treatment effect on the incidence of phytopathogenic fungi for the studied species/cultivars (Table 3). No cysts were found in any of the analyzed samples. In general, Cladosporium sp., Fusarium sp., Rhizoctonia sp., Meloidogyne sp., Filenchus sp., Aphelenchus sp., Aphelencoides sp., and Rotylenchulus sp. are the main plant phytopathogens associated with the seeds of the main tropical forage species used in integrated systems.

5.4 Discussion The water content may be a limiting factor for the germination of non-dormant seeds, affecting the percentage of germination, the germination rate and the uniformity of the process (Marcos Filho, 2015). The water content values found in experiment 1 (Table 1) are close to the range from 10.7 to 11.5 % obtained by Martins and Silva (2001); 10.39 % verified by Cardoso et al. (2014); and 10.5 % observed by Lima et al. (2014) in studies with forage seeds. However, in experiment 2, it was verified that the seeds had a water content below that found in the literature, but considered adequate (Table 1). Seeds with inadequate water contents may have problems related to the limitation of germination or deterioration due to damage caused by the deficiency or excess of water within the seeds (Câmara and Stacciarini-Seraphin, 2002; Marcos Filho, 2015). With regard to the first germination count, the Ruziziensis and Piatã grasses, being seeds of smaller size, compared to the remainder, present a larger surface exposed to contact with the substrate and, consequently, might have imbibed water and germinated more rapidly (Table 1) (Marcos Filho, 2015). According to Giurizatto et al. (2003), the thickness of the tegument interferes in the percentages of seed germination, although this parameter should not be used alone to define the physiological quality of the seeds. 94

The Ruziziensis grass presented higher GRI in both experiments (Table 1), which allows inferring that a great amount of seeds has the potential for a fast and uniform establishment, under field conditions (Pariz et al., 2010). It is probable that the higher GRI observed in the seeds of the cultivar Ruziziensis was driven by the lower dormancy of the seeds (Table 1), since dormancy influences the germination rate of the seeds, in an inversely proportional manner (Alves et al., 2017). It is also worth noting their greater vigor, seen from the results of the first germination count along with the results of the germination rate index and the germination of aged seeds (Table 1). Furthermore, as to the cultural value of the forages studied in both experiments (Table 1), it was observed that in the cases where the cultural value was obtained through the viability test, overestimated results were generated. The cultural value results based on seed viability do not fully meet the needs for crop implantation in the field (seed quantity per unit area) since this parameter does not consider the dormancy phenomenon. Dormancy is characterized as the non-germination of the seed even when subjected to favorable environmental conditions and is one of the main causes of low seed germination rates in forage species (Lacerda et al, 2010). Therefore, difficulties in the handling and problems such as reduced stand, a high percentage of invasive plants and low supply of forage to animals may arise due to an overestimated cultural value. This phenomenon occurred more frequently in the Xaraés and Marandu grasses (Table 1), probably due to differences in the chemical-physical constitution of its in tegument that hinders the gas exchange, hormonal balance, or embryo immaturity (Marcos Filho, 2015). According to Câmara and Stacciarini-Seraphin (2002), the germination inhibition of the Marandu grass by the seed coating possibly occurs due to a restriction to the gas exchanges. Corroborating this argument, Meschede et al. (2004) found that the removal of the wrapping in the Marandu grass seeds provided better germination results, indicating a strong tendency that the dormancy process in the cultivar has the coating as its main cause. 95

The seeds of the Xaraés and Marandu grasses probably have a differentiated thickness compared to the other species/cultivars, preventing larger proportions of gas exchange, which may culminate in high dormancy rates. However, further research is need on the chemical-physical constitution of the seed coat studied in this work. In addition, the dormancy phenomenon may be linked to the initial vigor of seed lots. In this sense, Trombeta (2013), when studying vigor tests associated with the overcoming of dormancy in seeds of Piatã grass observed that the lots that are more vigorous present lower index of the dormancy mechanism. This was valid for the Ruziziensis grass since it showed major vigor (first germination count, GRI, and germination of aged seeds) and low dormancy (Table 1). With regard to the percentage of germination after the accelerated aging, it was observed that the Xaraés and Marandu grasses had a lower germination percentage (Table 1). This probably occurs due to protein denaturation because of the high temperature to which they were subjected during aging. This phenomenon occurred only with these cultivars (Xaraés and Marandu grasses), possibly due to intrinsic characteristics of the seed lot, such as the tegument, as well as the embryo sensitivity to unfavorable environmental conditions, such as high temperature. In addition, they also presented lower vigor, as observed by the results of the first germination count in conjunction with the germination rate and the germination index of aged seeds (Table 1). In the sanitary analysis of Brachiaria seeds (Table 2), corroborating with the results found in this research, Mallmann et al. (2013), when studying the health of grass seeds produced in the Mato Grosso state, also reported the incidence of Cladosporium sp, Alternaria sp., and Fusarium sp. in seeds of Marandu grass, B. syn Urochloa sp. and Panicum syn. Megathyrsus maximum, respectively. Furthermore, Santos et al. (2014) and Sbalcheiro et al. (2014) also reported the incidence of Aspergillus niger, Fusarium sp., Penicillium sp. and Rhizopus sp. in seeds of Piatã grass. The fungi Cladosporium sp., Rhizoctonia sp., and Fusarium sp., which prevailed in this study (Table 2), can negatively impact the yield of the crop and consequently reduce the supply of food to the animals, since it causes a reduction in the germination 96

potential of the seeds (Marchi et al., 2007; Pedroso, 2009; Marchi et al., 2010; Sbalcheiro et al., 2014). Besides the physiological damages caused to the Brachiaria seeds, there are indications of the involvement of biotic agents in the mortality of the Marandu grass (MGM), among these, Pythium sp., Rhizoctonia sp., and Fusarium sp., especially in poorly-drained areas (Marchi et al., 2011). Therefore, it is recommended that lots of Marandu grass similar to the one analyzed in this study (both experiments) should not be implanted in areas with poor drainage since they have a high incidence of Rhizoctonia sp. and Fusarium sp. (Table 2). Although there was no treatment effect on the incidence of the fungus Rhizoctonia sp., the presence of this pathogen may cause leaf burning, collect rot and death in tropical grasses, soybean and maize. In cotton, it causes damping off, for example. In addition, it contributes to the reduction of vigor and germination rate of the seeds (Dias et al., 2013; Goulart, 2016; Poloni et al., 2016). Furthermore, the high frequency of Fusarium sp. observed in the studied lots may be linked to the ability by the fungi to produce resistance structures with high soil survival, culminating in the existence of alternative hosts such as cotton, Brachiaria, coffee, bean, maize and soybean (Barros and Juliatti, 2011). Fusarium sp. can cause seed necrosis and root rot in plants, reducing the booth (Almeida, 2015). Fusarium moniliforme and Fusarium graminearum, are notable for causing root and stem rot in infected plants (Costa et al., 2009); Fusarium clamydosporium is known for causing the wilt symptom followed by death in forage plants, such as Stylosanthes sp. (Verzignassi and Fernandes, 2001); and Fusarium oxysporum is notable for causing symptoms of wilt, yellowing, and death in Brachiaria plants (Almeida, 2015). Based on this, it is inferred that all lots within this study are unsuitable for single planting, intercropping, or crop rotation, and their adoption in integrated systems is highly compromised. With regard to the genus Cladosporium sp., the major barriers to the use of seeds contaminated with this fungus lie in the fact that some species may cause leaf spot and 97

lesions (Revankar and Sutton, 2010). They may have the ability to digest proteins in the epidermis, causing red spots or severe eruptions in animals and humans (Espinel-Ingroff et al., 1986). Therefore, the presence of this pathogen can cause problems in the implantation and conduction of the crop, as well as in animals and humans that have contact with areas of contaminated pastures. When analyzing the sanitary disinfestation process of the seeds, in the experiment 1, after the disinfestation process, it may be verified that the reduction of infestation by Fusarium sp., which is a fast-growing and aggressive fungus (Mallman et al., 2013) may have led to the growth of other fungi, such as Cladosporium sp. and Rhizoctonia sp. In addition, the increase in the frequency of the fungi Cladosporium sp. and Rhizoctonia sp. can be justified by the high incidence within the seed, conversely to Fusarium sp (high incidence on the seed surface) (Table 2). Conversely, in experiment 2, an incidence reduction trend was observed for all major fungi after the disinfestation process. This suggests a high incidence on the seed surface in these lots (Table 2). In the phytonematode analysis, the eggs and phytonematodes found by the method with incubation period may correspond to those that are present in the commercial sample, externally and internally transmitted with impurities to the seeds. The high incidence of both free-living and pathogenic phytonematodes after the incubation period may have occurred due to egg hatching and consequent emergence of juveniles, as well as the release of pathogens from inside of the seeds. The highest amount of eggs and phytonematodes is conveyed by the inside seeds. Mallmann et al. (2013) observed a high occurrence of Aphelenchoides sp. and Ditylenchus sp. in the Marandu and Piatã grasslands produced in the states of Mato Grosso and São Paulo. In addition, they found that Panicum syn. Megathyrsus maximum cultivars Massai and Mombaça presented a higher incidence of Aphelenchoides sp. and Ditylenchus sp., especially in the seeds produced in Mato Grosso. It is believed that phytonematodes can have a significant impact on the dry matter production and persistence of field forages (Favoreto, 2004). In addition, there is a confirmation of damage caused by the incidence of pathogenic phytonematodes in the 98

implantation of integrated systems, since exogenous pathogens can be inserted into areas. A phytonematode reported in this study, Meloidogyne sp., form galls in the roots, necrosis between the veins, small and yellow plants in reeds in areas producing soybean and maize (Freitas et al., 2001; Dias et al., 2010). Aphelencoides besseyi causes the Mad Soybean Disease II, besides attacking cotton, causing green stem and leaf retention (Dias et al., 2010; Favoreto et al., 2015; Meyer et. al., 2017). Rotylenchulus reniformis causes dwarfism, chlorosis and leaf yellowing in soybean and cotton crops (Embrapa Soja, 2010; 2013). In addition to direct damages, the phytonematodes constitute a major obstacle to the exportation of forage seeds. Large importers of Brachiaria syn. Urochloa sp. and Panicum syn. Megathyrsus sp. seeds impose phytosanitary restrictions on the Brazilian product (Marchi et al., 2007). Therefore, the use of seed of low sanitary quality causes the failure in the formation of pasture areas and the commercialization of seed lots (Mallmann et al., 2013), as well as in the large-scale adoption of more sustainable crop models, such as integrated systems.

5.5 Conclusions Cladosporium sp., Fusarium sp., Rhizoctonia sp., Meloidogyne sp., Filenchus sp., Aphelenchus sp., Aphelencoides sp., and Rotylenchulus sp. are the main phytopathogens associated with the seeds studied in this work. Comparing the seeds studied in this work, the seeds of ruziziensis grasses have better physiological quality and the seeds of xaraés grasses have better sanitary quality.

5.6 References Almeida MP. Quality of seeds of Brachiaria brizantha cv. Xaraés from the field of production to storage. Pelotas: Federal University of Pelotas, 2015.

Alves BA, Medeiros LT, Sales JF, Branquinho C, Silva JW, Souza RG. Germination of forage seeds of gender Brachiaria a function of environments and storage times. Global Science and Technology. 2017;10:11-19.

Balbino LC, Barcellos AO, Stone LF. Referencial Document Crop-Livestock-Forestry integration framework. Brasília: Embrapa, 2011. 99

Barros FC, Juliatti FC. Survey of fungi in samples received by micology and plants protect laboratory at Federal University of Uberlândia from 2001 to 2008. Bioscience Journal. 2012;8:77-86.

Brasil. Ministério da Agricultura, Pecuária e Abastecimento Manual of sanitary analysis of seeds. Ministry of Agriculture, Livestock and Food Supply. Brasília: NAD/ CGAL. 2009a.

Brasil. Ministério da Agricultura, Pecuária e Abastecimento. Rules for seed analysis. Ministry of Agriculture, Livestock and Food Supply. Brasília: MAPA/ACS. 2009b.

Brasil. Ministério da Agricultura. Normative Instruction Nº 30 of May 21, 2008. Norms and standards for production and commercialization of seeds of tropical forage species. 2008. Obtained from http://www.adagri.ce.gov.br/Docs/legislacao_vegetal/IN_30_de_21.05.2008.pdf. Accessed on: June 20, 2019.

Brasil. Ministério da Agricultura. Normative Instruction Nº 30 of October 26, 2010. Amendment of Normative Instruction Nº 30 of May 21, 2008. 2010. Obtained from http://www.in.gov.br/autenticidade.html. Accessed on: June 20, 2019.

Câmara HHLL, Stacciarini-Seraphin E. Germination of seeds of Brachiaria brizantha cv. Marandu under different storage periods and hormonal treatment. Pesquisa Agropecuária Tropical. 2002;32:21-28.

Coolen WA, D‟herde CJ. A method for the quantitative extraction of nematodes from plant tissue. Ghent: Nematology and Entomology Research Station, 1972.

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6. CONCLUSIONS (GENERAL) The cultivation of forages belonging to the genera Brachiaria syn. Urochloa sp. and Panicum syn. Megathyrsus sp., together with soybean, corn, sorghum and millet crops are prevalent in the ILP system of tropical regions. The phytomatomatoides Meloidogyne sp., Pratylenchus brachyurus, Heterodera glycines, Rotylenculus reniformis, Aphelenchoides sp., Ditylenchus sp. and Filenchus sp. can cause greater damage to the main cultures used in ILP. The main fungi reported as associated with forage and agricultural crops, and soils from integrated systems in tropical Brazilian regions are Bipolaris sp., Curvularia sp., Exserohilum syn. Helminthosporium sp., Phoma sp., Fusarium sp., Macrophomina sp., Pythium sp., Rhizoctonia sp. and Sclerotium sp. The increase in the contact time of the Brachiaria seeds treated with fertilizer reduces the incidence of fungi, improving the sanitary quality of the seeds. Cladosporium sp., Fusarium sp., Rhizoctonia sp., Meloidogyne sp., Filenchus sp., Aphelenchus sp., Aphelencoides sp., and Rotylenchulus sp. are the main plant phytopathogens associated with the seeds of the main tropical forage species used in integrated systems. Among the species studied, the Ruziziensis grass seeds present major physiological quality, whereas the Xaraés grass seeds present major sanitary quality. 104

APPENDIX Published articles 105

Journal of Experimental Agriculture International

37(4): 1-13, 2019; Article no.JEAI.49590 ISSN: 2457-0591 (Past name: American Journal of Experimental Agriculture, Past ISSN: 2231-0606)

Phytonematodes in Integrated Crop-livestock Systems of Tropical Regions

Anne Caroline Dallabrida Avelino1*, Dayana Aparecida de Faria1, Lucas Dias de Oliveira1, Bruno Giuseppe Terzi1, Alexandre Secco Contreras Filho1, Matheus Farinha Afonso1, Onassis Henrique Simon Rondon1, Giovani Oliveira de Arieira1, Joadil Gonçalves de Abreu1, Wender Mateus Peixoto1 and Mariana Rossi1

1Department of Agronomy and Zootechny, Federal University of Mato Grosso, Cuiabá, Mato Grosso, Brazil.

Authors’ contributions

This work was carried out in collaboration among all authors. Authors ACDA, GOA and JGA designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Authors ACDA, LDO and BGT managed the analyses of the study. Authors ACDA, DAF, LDO, BGT, ASCF, MFA, OHSR, JGA, WMP and MR managed the literature searches. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/JEAI/2019/v37i430275 Editor(s): (1) Dr. Daniele De Wrachien, Full Professor of Irrigation and Drainage, State University of Milan, Italy. Reviewers: (1) M. A. Mahmoud Youssef, National Research Centre, Egypt. (2) Liamngee Kator, Benue State University, Nigeria. Complete Peer review History: http://www.sdiarticle3.com/review-history/49590

Received 25 March 2019 Accepted 10 June 2019

Published 15 June 2019

ABSTRACT

*Corresponding author: E-mail: [email protected], [email protected]; 106

Journal of Experimental Agriculture International

39(5): 1-13, 2019; Article no.JEAI.50098 ISSN: 2457-0591 (Past name: American Journal of Experimental Agriculture, Past ISSN: 2231-0606)

Fungi Associated with Major Agricultural and Forage Crops in Integrated Systems of Brazilian Tropical Regions

Anne Caroline Dallabrida Avelino1*, Dayana Aparecida de Faria1, Lucas Dias de Oliveira1, Yuri Nunes Cervo1, Alexandre Secco Contreras Filho1, Matheus Afonso Farinha1, Onassis Henrique Simon Rondon1, Joadil Gonçalves de Abreu1, Wender Mateus Peixoto1, Mariana Rossi1 and Jéssica Rodrigues1

1Department of Agronomy and Zootechny, Federal University of Mato Grosso, Cuiabá, Mato Grosso, Brazil.

Authors’ contributions

This work was carried out in collaboration among all authors. Authors ACDA and JGA designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Authors ACDA, LDO, YNC and ASCF managed the analyses of the study. Authors ACDA, DAF, LDO, YNC, ASCF, MAF, OHSR, JGA, WMP, MR and JR managed the literature searches. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/JEAI/2019/v39i530343 Editor(s): (1) Dr. Francesco Montemurro, Professor, C. R. A. SSC - Research Unit of the Study of Cropping Systems, Metaponto, Italy. (2) Dr. Luis F. Goulao, Professor, Tropical Research Institute - IICT, Agri 4 Safe / Biotrop: Polo Mendes Hand, Agro-Industries and Tropical Agriculture Pavilion (3rd Floor), Portugal. (3) Dr. Lixiang Cao, Professor, Department of Biotechnology, Sun Yat-Sen University, China. (4) Dr. Vincenzo Tufarelli, Professor, Department of DETO - Section of Veterinary Science and Animal Production, University of Bari Aldo Moro, Italy. Reviewers: (1) Jaime Cuauhtemoc Negrete, Autonomous Agrarian Antonio Narro University, Mexico. (2) Cristiane Ramos Vieira, University of Cuiabá, Brazil. (3) Natalia Statsyuk, All-Russian Research Institute of Phytopathology, Russia. Complete Peer review History: http://www.sdiarticle3.com/review-history/50098

Received 12 May 2019 Accepted 22 July 2019

Published 30 July 2019

ABSTRACT

*Corresponding author: E-mail: [email protected], [email protected]; 107

Journal of Experimental Agriculture International

34(6): 1-9, 2019; Article no.JEAI.48521 ISSN: 2457-0591 (Past name: American Journal of Experimental Agriculture, Past ISSN: 2231-0606)

Effect of Fertilizer on Palisadegrass Seeds Pathogens Anne Caroline Dallabrida Avelino1*, Dayana Aparecida de Faria1, Dryelle Sifuentes Pallaoro1, Giovani Oliveira de Arieira1, Arthur Behling Neto2, Joadil Gonçalves de Abreu1, Elisangela Clarete Camili1, Leimi Kobayasti1 and Carlos Eduardo Avelino Cabral3

1Department of Agronomy and Zootechny, Federal University of Mato Grosso, Cuiabá, Mato Grosso, Brazil. 2Institute of Agrarian and Technological Sciences, Federal University of Mato Grosso, Sinop, Mato Grosso, Brazil. 3Institute of Agricultural Sciences and Technology, Federal University of Mato Grosso, Rondonópolis, Mato Grosso, Brazil.

Authors’ contributions

This work was carried out in collaboration among all authors. Author ACDA designed the study, performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscript. Authors DAF, DSP, GOA and ABN managed the analyses of the study. Authors JGA, ECC, LK and CEAC managed the literature searches. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/JEAI/2019/v34i630188 Editor(s): (1) Dr. Mohammad Reza Naroui Rad, Department of Horticulture Crops Research, Sistan Agricultural and Natural Resources Research and Education Center, Iran. Reviewers: (1) Mahmoud Abdelhamid Dawood, Kafrelsheikh University, Egypt. (2) Liamngee Kator, Benue State University, Nigeria. Complete Peer review History: http://www.sdiarticle3.com/review-history/48521

Received 02 February 2019 Accepted 11April 2019

Published 20 April 2019

ABSTRACT

108