Phytopathogenic agents associated with Plukenetia volubilis L. in Cuba: symptomatology and observations about their severity and degree of infestation

Plant Parasitic Nematodes

Plant Pathogenic Fungi

For identification of fungal pathogens, samples of 10 to 20 cm in length and diameters ranging from 1 to 10 cm were taken from stems and roots. In each case, the area of ​​growth of the damage was superficially disinfected with 1% sodium hypochlorite for 3 minutes and washed 3 times with abundant sterile distilled water (1414. Cabrera RI, Ferrer J, Peña I, Zamora V. Lasiodiplodia theobromae (Pat.) Griffon & Maubl. Sintomatología, afectaciones e impacto en la citricultura cubana actual. Levante Agrícola. 2012;51(412): 254-261. ).

Subsequently, tissue fragments of 3 to 4 mm² were removed with a scalpel and placed separately in Petri dishes. Plates (100 x 15 mm) were prepared with Potato Dextrose Agar (PDA) and H media (1414. Cabrera RI, Ferrer J, Peña I, Zamora V. Lasiodiplodia theobromae (Pat.) Griffon & Maubl. Sintomatología, afectaciones e impacto en la citricultura cubana actual. Levante Agrícola. 2012;51(412): 254-261. ), supplemented with oxytetracycline (0.4 mg. ml^-1), and incubated at 26 ± 2˚C in the dark. When the development of colonies of the fungus that predominated in the sowing points was observed, this fungal growth was transferred to test tubes containing the same medium for its conservation and study of the morphometric and morpho-cultural characteristics. The developed fungal structures were observed under a phase contrast microscope (640X). The fungi were identified by comparative sequence analysis, as well as by contrasting with the existing literature and using taxonomic keys (1515. Crous PW, Lombard L, Sandoval-Denis M, Seifert KA, Schroers HJ, Chaverri P., et al. Fusarium: more than a node or a foot-shaped basal cell. Studies in Mycol. 2021; 98:100116. doi:10.1016/j.simyco.2021.100116. eCollection Apr. PMID: 34466168., 1616. Marin-Felix Y, Groenewald J.Z, Cai LMCG, Chen Q, Marincowitz S, Barnes I. et al. Genera of phytopathogenic fungi: GOPHY 1. Studies in Mycol. 2017; 86:99-216. doi:10.1016/j.simyco.2017.04.002.).

Pathogenicity assay

Two isolates obtained from symptomatic Sacha Inchi plants, identified as Fusarium spp., were tested for pathogenicity for Sacha Inchi. The plants to be inoculated were washed thoroughly in sterile distilled water before being challenged with a concentrated spore suspension (1717. Pastrana AM, Kirkpatrick SC, Kong M, Broome JC,Gordon TR. Fusarium oxysporum f. sp. mori, a new forma specialis causing Fusarium wilt of blackberry. Plant Disease. 2017; 101:2066-2072. https://doi.org/10.1094/PDIS-03-17-0428-RE ). The spore suspension was prepared from fully colonized plates of PDA and adjusted to a density of 10⁶ spores.ml^-1 in sterile water. Control plants were treated with sterile water instead of a spore suspension. Inoculated and control plants were left at temperatures ranging from 27 to 30°C and relative humidity around 75%. Two weeks later, the plants were rated on a semicontinuous 1 to 5 scale, where: 1 = no symptoms; 2 = drying and death of older leaves; 3 =symptomatic older leaves accompanied by wilting of younger leaves; 4 = severe foliar symptoms; 5 = dead plant.

Plant Parasitic Nematodes (PPN)

Nine species of plant parasitic nematodes were found in the sampled areas (Table 2). The endoparasitic root-knot nematodes Meloidogyne arenaria (Neal) Chitwood and Meloidigyne incognita (Kofoid & White) Chitwood are considered among the most important nematodes worldwide (1818. Sikora RA, Coyne D, Hallmann J, Timper P. Reflections and Challenges Nematology. Nematode parasites of banana and plantains. pp. 1-21. In: Sikora RA, Coyne D, Hallmann J. Timper P. (eds.). Plant parasitic nematodes in subtropical and tropical agriculture. 3^rd. Edition. CABI International and USDA. Boston. MA. 2018. 876 pp.). Similar results to those previously reported (1919. Wang Y, Xie Y, Cui HD, Dong Y. First Report of Meloidogyne javanica on Sacha Inchi (Plukenetia volubilis) in China. Plant Disease. 2014; 98 (1): 165., 2020. Márquez-Dávila K, Cayotopa J, Arévalo L, Vivanco U, Arévalo J. Diagnóstico y niveles de inóculo del nematodo que afecta a la raíz de Sacha Inchi (Plukenetia volubilis L.) en Perú. Fitopatología. 2007; 02 (42): 52 - 53.) were observed in all the sampled sites; they were associated with deterioration of necks and roots of plants and the presence of knot- shaped galls. As mentioned above, Rodríguez & Hernández-Ochandia (55. Rodríguez MG, Hernández-Ochandía D. Nuevos hospedantes de Meloidogyne spp. para Cuba. Rev. Protección Veg. 2020; 35 (1): E-ISSN:2224-4697) had already reported the presence of Meloidogyne arenaria in Cuban Sacha Inchi plantations.

The three sampled’ sites showed heavy infestations of mainly M. incognita in soil and roots (4 to 5 grades of infestation), deterioration of adult plants, decreased production, the presence of knot-shaped galls, loss of secondary roots, necrosis, and neck rot. These results agreed with other authors (2121. Manzanilla RH, Evans K, Bridge J. Plant diseases caused by nematodes. In: ZX Chen, SY Chen & DW Dickson (eds.) Vol. II: Nematode management and utilization. Nematology Advances and Perspectives. Tsinghua Univ. Press.; CABI Publishing. Wallingford. UK; 2004; 1219pp., 2222. Fernández-Gonzálvez E, Gandarilla-Basterrechea H, Almarales-Antúnez M, Almandoz-Parrado J, Rodríguez-Hernández M. Determinación y sintomatología de Xiphinema basiri Sidiqqi en chile habanero (Capsicum chinense Jacq. en Cuba. Rev. Protección Veg. 2022; 37 (2): e-ISSN 2224-4697. https://cu.id.com/2247/v37n2e12 +), who also reported poor plant growth, foliage yellowing, low production and decreased fruit quality in Sacha Inchi.

The phytosanitary diagnosis of the crop showed deterioration of the neck and roots of the adult plants and the presence of knot-shaped galls, which confirmed the existence of nematodes. Several of these results agree with those previously reported (2222. Fernández-Gonzálvez E, Gandarilla-Basterrechea H, Almarales-Antúnez M, Almandoz-Parrado J, Rodríguez-Hernández M. Determinación y sintomatología de Xiphinema basiri Sidiqqi en chile habanero (Capsicum chinense Jacq. en Cuba. Rev. Protección Veg. 2022; 37 (2): e-ISSN 2224-4697. https://cu.id.com/2247/v37n2e12 +, 2323. Caboni P, Aissani N, Demurtas M, Ntalli N, Onnis V. Nematicidal activity of acetophenones and chalcones against Meloidogyne incognita, and structure- activity considerations. Pest Manag. Sci. 2016; 72, 125-130.). The most complex situation was at the UBP “50th Anniversary of the Victory of Playa Girón”. Six species of parasitic nematodes were found in the soil and root samples from the “50 Anniversary”.

In general, the root-knot nematodes M. incognita and M. arenaria were the species that showed the highest population levels and those most strongly associated with root damage.

In the case of the UBP "El Pitirre", five species of parasitic nematodes were found in the soil and root samples. The nematodes M. incognita and M. arenaria once again presented the highest population levels and were strongly associated with root damage. The ectoparasite Xiphinema spp. was detected only in this UBP, with a medium level population in a plantation and a low population in other. This species can also damage other crops and should be further assessed, since its symptoms on the roots can be confused with those caused by Meloidogyne sp. Due to the importance of the other two species determined (R. reniformis and Criconemoides spp.) as causal agents of damage on other crops, they should also continue to be evaluated.

In the case of the UBP "Nazareno", three species of parasitic nematodes were found in the soil and root samples. The nematodes M. incognita and M. arenaria once again presented the highest population levels and were closely associated with root damage. This marked a serious deterioration of the entire root system.

The root knot nematodes M. incognita and M. arenaria appeared in all locations with a significant level of attack. Nevertheless, in spite of these findings, these results will be confirmed by molecular analysis based on PCR amplification.

According to table 1, plantain was the previous crop planted in UBPs “50 Anniversary” and “Nazareno”. This crop is highly susceptible to nematodes, which may justify the high incidence of these plagues. However, the previous crop planted in the UBP “El Pitirre” was Tithonia diversifolia H.W. Schott. No reference on susceptibility level of Tithonia to nematodes was found. Ramírez-Pedroso (Pers. Communication) evaluated indicators, such as fresh and dry matter, botanic seed production, flowering density, and yield of T. diversifolia associated with Cannavalia eusiformis de Geer and did not observe any effect due to nematodes or the presence of galls.

The phytosanitary diagnosis carried out in the three UBPs of the CIPB showed deterioration of adult plants, decreased production, the presence of knot-shaped galls, loss of secondary roots, necrosis and neck rot. These results agreed with those already reported (2020. Márquez-Dávila K, Cayotopa J, Arévalo L, Vivanco U, Arévalo J. Diagnóstico y niveles de inóculo del nematodo que afecta a la raíz de Sacha Inchi (Plukenetia volubilis L.) en Perú. Fitopatología. 2007; 02 (42): 52 - 53.), which also included low plant growth, foliage yellowing, low production, and decreased fruit quality in the Sacha Inchi crop studied.

It is convenient to mention that these are the three most important farms of Sacha Inchi production in Cuba because of the extension of land planted with this crop.

Although in all cases the cultivar Inca-1 was used, the damage caused by the presence of nematodes, particularly Meloidogyne spp., have been reported to depend on the plant cultivar (33. Márquez-Dávila K, R Gonzales, L Arévalo, R Solís. Respuesta de accesiones de sacha inchi (Plukenetia volubilis L.) a la infestación inducida del nematodo Meloidogyne incognita (Kofoid & White, 1919) Chitwood, 1949. Folia Amazónica. 2013; 22 (1-2): 17 97-103.). In other works, it was also mentioned that the growth rate of plants and the number of nodules per root were directly proportional to the level of infestation (2020. Márquez-Dávila K, Cayotopa J, Arévalo L, Vivanco U, Arévalo J. Diagnóstico y niveles de inóculo del nematodo que afecta a la raíz de Sacha Inchi (Plukenetia volubilis L.) en Perú. Fitopatología. 2007; 02 (42): 52 - 53., 2121. Manzanilla RH, Evans K, Bridge J. Plant diseases caused by nematodes. In: ZX Chen, SY Chen & DW Dickson (eds.) Vol. II: Nematode management and utilization. Nematology Advances and Perspectives. Tsinghua Univ. Press.; CABI Publishing. Wallingford. UK; 2004; 1219pp.). Cultivar Inca-1 planted in the three UBPs, like many of the cultivated accessions of Sacha Inchi, has a typical behavior as susceptible to Meloidogyne spp., with the consequence that productivity diminishes and the plants die in a few years after plantation (2020. Márquez-Dávila K, Cayotopa J, Arévalo L, Vivanco U, Arévalo J. Diagnóstico y niveles de inóculo del nematodo que afecta a la raíz de Sacha Inchi (Plukenetia volubilis L.) en Perú. Fitopatología. 2007; 02 (42): 52 - 53.).

Xiphinema sp. is an ectoparasitic species with economic importance in different crops (2222. Fernández-Gonzálvez E, Gandarilla-Basterrechea H, Almarales-Antúnez M, Almandoz-Parrado J, Rodríguez-Hernández M. Determinación y sintomatología de Xiphinema basiri Sidiqqi en chile habanero (Capsicum chinense Jacq. en Cuba. Rev. Protección Veg. 2022; 37 (2): e-ISSN 2224-4697. https://cu.id.com/2247/v37n2e12 +). High populations of this plant-parasitic nematode (more than 150 nematodes per 100 g of soil) were detected only in UBP “El Pitirre”. It was associated with the presence of big rounded and chained galls on the roots. Because of its importance for many crops and the relative similarity of its symptoms on roots with those by Meloidogyne spp., which can be confused with it (2323. Caboni P, Aissani N, Demurtas M, Ntalli N, Onnis V. Nematicidal activity of acetophenones and chalcones against Meloidogyne incognita, and structure- activity considerations. Pest Manag. Sci. 2016; 72, 125-130.), studies about its distribution and pathogenicity must be continued.

Low populations of other species, such as the ectoparasitic nematodes A. bicaudatus, Criconemoides spp., Trichodorus spp., Tylenchorhynchus spp., and the semi-endoparasitic R. reniformis, were also found, and they will continue to be followed because of their importance in some other crops. All the reports of the nematode species, including M. incognita, in this paper are new for this crop in Cuba.

Additionally, the wounds caused on plant roots by plant-parasitic nematodes also act as entry routes for secondary pests and pathogens (2323. Caboni P, Aissani N, Demurtas M, Ntalli N, Onnis V. Nematicidal activity of acetophenones and chalcones against Meloidogyne incognita, and structure- activity considerations. Pest Manag. Sci. 2016; 72, 125-130.).

Plant Pathogenic Fungi

Regarding the phytopathogenic fungi, the symptoms of neck rot on Sacha Inchi seedlings pointed to the presence of Fusarium spp. (Fig. 2A). Other symptoms detected in the plantations were dry branches (Fig. 2B) and necrosis of xylem in main branches (Fig. 2C and D). Necrosis of xylem, as well as sectorial death in branches, pointed to the presence of the fungus Lasiodiplodia sp.

Figures 3A and B showed the presence of fruiting bodies of this phytopathogenic fungus. Typical conidia of this genus were also observed (Fig. 3C).

By observations under the optical microscope, microconidia and typical conidia of the genus Fusarium sp. were revealed from the mycelium produced when the fungus present on stems and branches was grown in humid chamber (Fig. 4A and B). The observed phytosanitary damages agree with those reported by Acosta- Ramírez (2424. Acosta-Ramírez CJ. Caracterización fisiológica y fitosanitaria de las semillas de Sacha Inchi (Euphorbiaceae: Plukenetia volubilis). Universidad del Valle. Facultad de Ciencias. [Thesis of Magister in Sciences - Biology]. 2018. Santiago de Cali. [Online]. Available in: http://www.repositoriounivalle.edu.co/licencia/2020 ), who reported the presence of these two fungi besides other fungal genera and species not found in this work. Other fruiting bodies were observed, but these fungi have not yet been identified. The presence of these two fungi are also new reports for the crop in Cuba. In the case of Lasiodiplodia spp., it could be the first report not only for Cuba. Our request allowed finding only a previous report for the rest of Sacha Inchi producing countries (2424. Acosta-Ramírez CJ. Caracterización fisiológica y fitosanitaria de las semillas de Sacha Inchi (Euphorbiaceae: Plukenetia volubilis). Universidad del Valle. Facultad de Ciencias. [Thesis of Magister in Sciences - Biology]. 2018. Santiago de Cali. [Online]. Available in: http://www.repositoriounivalle.edu.co/licencia/2020 ). In order to identify these fungal species, their molecular characterization is in progress by means of PCR amplification using highly- conserved sequences, such as Internal transcribed spacers (ITS) region, Translation and elongation factor (TEF-1α) coding region or β-tubulin genes.

It is important to mention that the presence of fungi is expected once the nematode causes a root wound, since it opens a gateway for fungi, which increase the potential of destruction and accelerate the plant death (2525. Pinochet JG, Stover RH. Fungi in lesions caused by burrowing nematodes on bananas and their root and rhizome rotting potential. Trop. Agric. (Trinidad). 1980; 57 (3): 227-232.).

Pathogenicity assay

Concerning the pathogenicity test, the artificial inoculations allowed identifying the damage caused by the fungus Fusarium sp. on the Sacha Inchi seedlings (Fig. 4C). The affected tissue can be distinguished by comparing the treated plant with the control one, which does not show any signs of involvement. The Sacha Inchi seedlings inoculated with the assayed isolates developed symptoms that ranged between 3 and 4 into the 1 to 5 scale. That means symptomatic older leaves accompanied by wilting of younger leaves to severe foliar symptoms.

The diagnosis also made it possible to detect the presence of the Meloidogyne-Fusarium complex, with the majority presence of the root knot nematode Meloidogyne incognita. As previously mentioned, the presence of typical microconidia and conidia of the genus Fusarium spp. were observed (Figure 4C).

The Meloidogyne-Fusarium complex, which was found to affect these plants, adds a further complication to the sanitary situation. This complex has been reported to cause serious damage to Sacha Inchi plantations (66. Guerrero-Abad JC, Padilla-Domínguez A, Torres-Flores E, et al. A pathogen complex between the root knot nematode Meloidogyne incognita and Fusarium verticillioides results in extreme mortality of the inka nut (Plukenetia volubilis). J. Applied Botany and Food Quality. 2021; 94: 162-168., 1919. Wang Y, Xie Y, Cui HD, Dong Y. First Report of Meloidogyne javanica on Sacha Inchi (Plukenetia volubilis) in China. Plant Disease. 2014; 98 (1): 165.). The combination of both agents reinforces their effect because the wounds caused by the nematodes serve as entry routes for soil-borne plant phytopathogenic fungi l, particularly such as those of the Fusarium genus (2626. Agrios GN. Plant Pathology. 5th ed. Elsevier. Academic Press, New York. 2005; 921 pp.). The combined effect of these two phytopathogenic agents is far more destructive than when they act separately. It drastically reduces the yield and lifespan of this crop (33. Márquez-Dávila K, R Gonzales, L Arévalo, R Solís. Respuesta de accesiones de sacha inchi (Plukenetia volubilis L.) a la infestación inducida del nematodo Meloidogyne incognita (Kofoid & White, 1919) Chitwood, 1949. Folia Amazónica. 2013; 22 (1-2): 17 97-103.). This is the first study reporting Fusarium spp., Lasiodiplodia spp. and the Meloidogyne-Fusarium complex in Cuba.

The phytopathogens detected in this work are also important in different crops and, given the observed effects, it was recommended to deepen these studies and apply management measures compatible with the crop.