Research

PEREIRA, MVM; ALEXANDRE, MAV; CHAVES, ALR; SOUZA, ACO; BERNACCI, LC; NAGAI, A; SANTOS, DYAC; DUARTE, LML. 2021. Endemic Atlantic Forest species of as inhibitors of viral infection in zucchini. Horticultura Brasileira 39:146-154. DOI: http://dx.doi.org/10.1590/ s0102-0536-20210204 Endemic Atlantic Forest species of Caryophyllales as inhibitors of viral infection in zucchini Marcos Vinicius M Pereira 1 ID; Maria Amélia V Alexandre 1 ID; Alexandre Levi R Chaves 1 ID; Ana Cláudia O de Souza 2 ID; Luís Carlos Bernacci 2 ID; Alice Nagai 3 ID; Déborah Yara AC dos Santos 3 ID; Lígia Maria L Duarte 1 ID 1Instituto Biológico (IB), São Paulo-SP, Brasil; [email protected]; [email protected]; [email protected]; ligia. [email protected]; 2Instituto Agronômico de Campinas (IAC), Campinas-SP, Brasil; [email protected]; [email protected]; 3Universidade de São Paulo (USP), São Paulo-SP, Brasil; [email protected]; [email protected]

ABSTRACT RESUMO In Brazil, zucchini (Cucurbita pepo) is a socioeconomically Espécies de Caryophyllales endêmicas da Mata Atlântica important vegetable affected by damage caused primarily by zucchini como inibidoras de infecção viral em abobrinha-de-moita yellow mosaic virus (ZYMV). Although the occurrence of cucumber No Brasil, a abobrinha-de-moita (Cucurbita pepo L.) é uma mosaic virus (CMV) is less frequent, in C. pepo ‘Caserta’ it olerícola de importância socioeconômica que tem sofrido prejuízos can cause symptoms such as mottle, mosaic, leaf and fruit distortion, crescentes, devido principalmente aos danos causados pela infecção as well as reduced development. To minimize the damage, the pelo zucchini yellow mosaic virus (ZYMV). Embora a ocorrência do most widely used management technique is the preventive, albeit cucumber mosaic virus (CMV) seja menos frequente, em plantas de C. inefficient, application of insecticides, aimed at controlling aphids, pepo ‘Caserta’ ele pode causar sintomas como mosqueado, mosaico, the vectors of this virus. Thus, the search for more effective and distorção de folhas e frutos, além de redução do desenvolvimento das less environmentally harmful control methods has been the target plantas. Para minimizar o prejuízo, a medida de manejo mais utilizada of investigations. The purpose of the present study was to assess é a aplicação preventiva, porém ineficiente, de inseticidas, visando the action of the extracts of four native Caryophyllales species, as ao controle da população de afídeos, vetores desses vírus. Assim, a inhibitors of infection by CMV and ZYMV in C. pepo, in addition busca por formas de controle ambientalmente menos danosas tem to evaluating the possible induced resistance in this species. sido alvo de investigação. Este estudo visou avaliar a ação de extratos Fresh leaf extracts (LEs) of Guapira opposita, Pisonia ambigua foliares de espécies nativas de Caryophyllales, como inibidoras (), Gallesia integrifolia and Seguieria langsdorffii da infecção pelo CMV e ZYMV em C. pepo e como indutoras de (), previously assessed in the resistência. Extratos de folhas frescas (EFs) de Guapira opposita, / glutinosa pathosystem, were submitted to progressive Pisonia ambigua (Nyctaginaceae), Gallesia integrifolia e Seguieria dilutions sprayed on cotyledonary C. pepo leaves 30 min before langsdorffii (Phytolaccaceae), previamente avaliados no patossistema inoculation with CMV and ZYMV. Leaf extracts of G. integrifolia tobacco mosaic virus / Nicotiana glutinosa, foram submetidos a did not induce inhibition in any of the pathosystems assessed. diluições progressivas e pulverizados em folhas cotiledonares de Guapira opposita LEs inhibited the infection of plants inoculated C. pepo, 30 min antes da inoculação com o CMV e ZYMV. EFs with ZYMV below 50% but inhibited CMV infection by 70% at a de G. integrifolia não induziram qualquer inibição em nenhum dos concentration of 1:40. Given that leaf extracts of P. ambigua and S. patossistemas. EFs de G. opposita induziram inibição da infecção das langsdorffiiinduced high percentage inhibition, evident in the number plantas inoculadas com o ZYMV inferior a 50%. Na concentração of asymptomatic plants and confirmed by serological tests, these de 1:40, inibiu a infecção das plantas pelo CMV em 70%. Como species were selected to assess induced resistance in pre-treatment os extratos foliares de P. ambigua e S. langsdorffii induziram altas experiments. The LEs were efficient in inhibiting ZYMV and CMV porcentagens de inibição, essas espécies foram selecionadas para infection in C. pepo when applied up to 48 h before inoculation. The avaliação de indução de resistência, em experimentos de pré- LEs of S. langsdorffii and G. opposita, also tested for this system, tratamentos. Os EFs inibiram a infecção de ZYMV e CMV em C. were efficient when applied up to 72 h before CMV inoculation. pepo, quando aplicados até 48 h antes da inoculação. Os EFs de S. The LEs can be prepared from dry leaves and maintained at -20°C langsdorffiie G. opposita, também testada para esse sistema, foram for at least three years, conserving their inhibitory activity. These eficientes quando aplicados até 72 h antes da inoculação do CMV. results expand the possibilities for producers and consumers alike Constatou-se que os EFs podem ser preparados a partir de folhas in the sustainable management of the main zucchini viruses, without secas e mantidos a -20°C por, pelo menos três anos, conservando a damaging the environment. sua atividade inibidora. Esses resultados expandem as possibilidades de manejo das principais viroses da abobrinha-de-moita, de maneira sustentável, sem causar danos ao ambiente, aos produtores e aos consumidores.

Keywords: Cucurbita pepo, Cucumovirus, Potyvirus, alternative Palavras-chave: Cucurbita pepo, Cucumovirus, Potyvirus, controle control, antiviral. alternativo, antiviral. Received on September 21, 2020; accepted on April 6, 2021

146 Horticultura Brasileira 39 (2) April - June, 2021 Endemic Atlantic Forest species of Caryophyllales as inhibitors of viral infection in zucchini

ccording to a Brazilian Federal which are often recommended as distortion (Lima, 2011). AOfficial Gazette (DOU) publication, an insect/vector control strategy to Given that insecticide application among the 474 agrochemicals authorized prevent virus infections. This control has proved inefficient in controlling for commercialization, only 40 (8.4%) strategy, however, produces adverse ZYMV and CMV transmission by aphids are classified as biological and legally environmental effects and has been (Eigenbrode et al., 2018), alternative sanctioned as organic agricultural inputs increasingly questioned (Castle et al., methods such as the application of plant (DOU, Act No. 82, November 25, 2009). Unfavorable environmental extracts with inhibitory action against 2019). Given this scenario, Brazil and conditions, in addition to viral diseases viral infection may be a sustainable other countries that import Brazilian and the lack of resistant cultivars, strategy for the zucchini (C. pepo) agricultural products find themselves at have negatively influenced zucchini cultivation. This strategy can minimize a serious impasse since the population production, discouraging farmers from the damage caused by the virus, protects is increasingly consuming healthier maintaining production (Lecoq & Katis, producer health, prevent environmental foods and prioritizing less industrialized 2014). waste, and enable the production products with as few agrochemicals Among the ten virus species reported of healthy foods. In this scenario, as possible. Following this trend, the in cucurbits that can affect Brazilian studies conducted with leaf extracts of United Nations (UN) declared 2020 as zucchini production are zucchini Caryophyllales species have produced the international year of plant health, yellow mosaic virus (ZYMV, family promising results. making agribusiness increasingly Potyviridae, genus Potyvirus) and visible and inducing producers to adopt cucumber mosaic virus (CMV, family Caryophyllales is one of the largest alternative pest and disease control Bromoviridae, genus Cucumovirus) orders of (APG IV, 2016), with techniques, to guarantee phytosanitary (Lima & Alves, 2011; ICTV, 2017). The approximately 12,500 species distributed and food security, thereby meeting the ZYMV and CMV are non-persistently in 39 families (Hernández-Ledesman et demands of the internal and external transmitted by around 60 aphid species, al., 2015), including Nyctaginaceae consumer market (UN, 2020). which means that the viral particles are and Phytolaccaceae, which contain Despite UN recommendations acquired and transmitted in seconds or species with widely-studied inhibitory (2020), Brazil is one of the world’s minutes, during bite tests, adhering to activity against viral infection due to leading consumers of agrochemicals. the inner tip of the sucking apparatus antiviral proteins (Grasso & Shepherd, São Paulo state reported the highest of aphids (stylets). As such, insecticide 1978; Duarte et al., 2021). In addition number of poisonings over the last application is not an efficient strategy to spectabilis and ten years, totaling more than 8,000 to prevent viral dispersion in this crop Phytolacca thyrsiflora, which exhibited cases (Tavares et al., 2020). One of (Eigenbrode et al., 2018). excellent results (Noronha et al., 1989; the consequences of this excessive use ZYMV is restricted to cucurbits Duarte et al., 1990), viral inhibitors have is reflected in the induced resistance and was described in Citrullus lanatus been extensively studied only in Celosia of insects and phytopathogens as well (watermelon, Cucurbitaceae), in São argentea (), Dianthus as the high residual concentrations of Paulo state, in the early 1990s (Pavan caryophyllus (Caryophyllaceae), insecticide and fungicide molecules et al., 2016). The virus has been Boerhaavia difusa, Mirabilis jalapa in the final product (Pedlowski et al., increasingly reported in all cucurbit and B. spectabilis (Nyctaginaceae) and 2012). This has prompted debate and producing regions of Brazil (Yuki et al., Phytolacca americana (Phytolaccaceae) studies among specialists who warn of 2000; Lima, 2011; Spadotti et al., 2015). (Awasthi et al., 2015). Tozetto et al. the harmful effects of these molecules Intense yellowing between the veins, (2002) described 60% inhibitory action on health and the environment (Mata, mosaic pattern, blistering and extremely of Mirabilis jalapa (Nyctaginaceae) 2013; Starling et al., 2019). reduced and deformed leaf blades are leaf extract in C. pepo ‘Caserta” / The situation is worrisome, primarily symptoms that emerge after infection, in the vegetable segment, since most of causing the plant to atrophy. Early ZYMV pathosystem when applied up the foods produced (leaves, flowers, and infection in the zucchini interferes in to 120 h before virus inoculation, and fruits) are consumed fresh or minimally flowering and fruit production (Pavanet virus-infection inducible resistance has processed (Sediyama et al., 2014). al., 2016). In addition, fruit deformations been associated with these extracts. Zucchini (Cucurbita pepo, may cause damage during production Elbeshehy (2017) found 90% inhibition Cucurbitaceae), one of the ten most and compromise seed quality. of ZYMV in watermelon (Citrullus widely produced seed-propagated crops Despite infecting several species of lanatus, Cucurbitaceae) treated with in Brazil, stands out for its considerable plants and spontaneous vegetation, the LEs from B. spectabilis 48 h before socioeconomic importance yielding incidence of CMV is lower than ZYMV inoculation, while Tozetto et al. (2002) 65,711.4 t in 39 municipalities in São in cucurbits (Pavan et al., 2016). In demonstrated inhibitory activity of M. Paulo state, encompassing an area of general, CMV induces mottle, mosaic jalapa LE sprayed up to 120 h before 4,125.71 ha (IEA, 2019). Unfortunately, and leaf distortion symptoms in C. pepo ZYMV inoculation in C. pepo ‘Caserta’. this crop is also noteworthy for the ‘Caserta’ plants, as well as yellowing, Bharathi (1999) showed that M. jalapa intensive application of insecticides, reduced plant development and fruit extract inhibits CMV infection in Horticultura Brasileira 39 (2) April - June, 2021 147 MVM Pereira et al.

Solanum melongena (eggplant) by 81, MATERIAL AND METHODS controls, 50 µL of the purified virus (100 64 and 44%, when applied 72, 96 and µg/mL) were applied to the right half. 120 h before inoculation, respectively. After local necrotic lesions appeared, Extracts obtained from several Species collection and leaf extract they were counted to determine the (LE) preparation species of Caryophyllales were also percentage inhibition (PI), calculated efficient in reducing local symptoms The investigation and species based on the formula: 100 − (T/C x 100), caused by the following viruses: collection of G. integrifolia, G. where T = number of lesions on the left Amaranthaceae and Basellaceae / opposita, P. ambigua, and S. langsdorffii (treatment) and C = number of lesions (Figure 1) were conducted based on tobacco necrosis virus (TNV) / on the right (controls). IAC Herbarium data (http://herbario. Obtaining viral isolates and Phaseolus vulgaris (Smookler, 1971), iac.sp.gov.br), with records from the inoculum preparation Amaranthaceae, Nyctaginaceae, municipality of Campinas, São Paulo Isolates used for viral infection and Talinaceae / TMV state. Two Atlantic Forest fragments inhibition tests in a virus x systemic / N. glutinosa (Noronha et al., 1980, from the semideciduous seasonal host system, previously identified and 1983), Caryophyllaceae / TNV / P. forest physiognomy were selected for characterized, are part of the collection vulgaris (Barakat & Stevens, 1980), and sampling effort, namely Mata Santa of the Laboratório de Fitovirologia e Amaranthaceae / TMV / N. glutinosa Elisa (22°51’S, 47°04’W) and Bosque Fisiopatologia Karl M. Silberschmidt, (Choi & Jung, 1984). dos Jequitibás (22°55’S, 47°03’W). Instituto Biológico. ZYMV and CMV In the Atlantic Forest, an important Exsiccates that registered species isolates were obtained from naturally biome in São Paulo state, Brazil (Ribeiro occurrence in the area were deposited infected Lagenaria siceraria (calabash) in the IAC herbarium, under numbers et al., 2009), several Caryophyllales and Melothria pendula (creeping 46457 to 46459 (G. opposita), 46560 cucumber) plants, respectively, families can be found. However, few (G. integrifolia), 36471 and 46559 (P. intercropped with Cucurbita pepo studies on these native species as ambigua) and 56571 and 56722 (S. ‘Caserta’ (zucchini) in the municipality inhibitors of viral infection have been langsdorffii). of Monte Alegre do Sul-SP (22°40’55”S, conducted. Excellent results with The extracts were prepared from 1 46°40’51”W). To conduct the inhibition B. spectabilis (Nyctaginaceae) and g fresh leaves ground with 5 mL 0.01M tests, the viral inocula were standardized Phytolacca thyrsiflora(Phytolaccaceae) phosphate buffer, pH 7.0, added with at a concentration (w/v) of 1 g infected (Noronha et al., 1989; Duarte et al., 0.5% sodium sulfite (inoculation buffer leaves per 3 mL of IB. 1990) have been reported. As such, - IB), using an ultra-turrax homogenizer. other native species belonging to these The crude extracts were filtered through Determination of the highest families should be studied in depth as gauze. dilution with leaf extract (LE) activity potential alternatives in the control of The crude extracts, prepared from Preliminary assessment of the the leaves of four Caryophyllales plant viruses affecting economically viral inhibition effect using the half species, were submitted to progressive important crops, aimed at sustainable and leaf test dilutions up to 1:1,280 (v/v), for the eco-friendly production. In this context, In order to assess the viral inhibition ZYMV / Cucurbita pepo pathosystem the main goal of the present study was potential of aqueous extracts in a and 1:640 (v/v), for CMV / C. pepo to assess the efficacy of leaf extracts virus x local host system, the half with IB. from the species Guapira opposita leaf experiment was conducted by Cucurbita pepo ‘Caserta’ seedlings, and Pisonia ambigua (Nyctaginaceae) mechanically inoculating the indicator obtained after planting, were used for and Gallesia integrifolia and Seguieria species Nicotiana glutinosa () the test. The different extract dilutions langsdorffii (Phytolaccaceae), all with viral isolate from tobacco mosaic were sprayed on the cotyledonary endemic to the Atlantic Forest, as virus (TMV, Tobamovirus). This system leaves and after 30 min, 30 µL of viral inhibitors of infections caused by was used because N. glutinosa plants inoculum were applied, using 10 plants develop a hypersensitivity reaction CMV and ZYMV in Cucurbita pepo for each dilution. The cotyledonary when infected with TMV isolates, ‘Caserta’. It should be noted that ZYMV leaves of 10 control plants were applied inducing local necrotic lesions. To was selected for inhibition tests, since with 30 µL of inoculum. The plants perform the test, the aqueous extracts it is one of the potyviruses that cause with visible systemic symptoms were (1:5 dilution, w/v) of the four species counted fifteen days after inoculation in the most damage to zucchini culture were added with an equal volume of order to establish percentage inhibition, in Brazil. Although the occurrence of purified TMV solution (200 µg/mL), in calculated based on the formula: 100 the CMV is less frequent, in C. pepo order to obtain a final inhibitor extract − (T/C x 100), where T = number of ‘Caserta’ plants it can cause severe concentration of 1:10 (v/v) and 100 symptomatic plants treated with LEs, symptoms. In addition, CMV is also a µg/mL of purified solution of the virus and C = the number of symptomatic model for understanding plant – virus (treatment). Fifty µL aliquots of leaf control plants. In addition, leaf samples interactions (Lima, 2011; Jacquemond, extract + TMV were rubbed on the from treated plants and controls were 2012). left half of the N. glutinosa leaf. For collected for subsequent serological 148 Horticultura Brasileira 39 (2) April - June, 2021 Endemic Atlantic Forest species of Caryophyllales as inhibitors of viral infection in zucchini

testing. treatments, analyses were carried out from the four native species used in this Assessment of induced resistance in the symptomatic and asymptomatic study, with values above 70% (Table 1). in C. pepo C. pepo leaf extracts, as well as those Efficient reduction of local symptoms With a view to assessing induced of control plants. ZYMV was detected has been described for several extracts resistance in C. pepo, the LEs from by Double Antibody Sandwich - of Caryophyllales species (Smookler, the species exhibiting the greatest viral ELISA (DAS-ELISA), according to the 1971; Noronha et al., 1980, 1983; inhibition potential were sprayed on the manufacturer’s protocol (AGDIA®) and Barakat & Stevens, 1980; Choi & Jung, cotyledonary leaves of 10 C. pepo plants CMV by Plate Trapped Antigen – ELISA 1984). 24, 48, 72 and 96 h before mechanical (PTA-ELISA) with antiserum belonging Due to the high efficiency observed inoculation with 30 µL of ZYMV and to the serum bank of the Laboratório de in the local system, the LEs from the CMV isolates at a concentration of 1:3 Fitovirologia e Fisiopatologia, Instituto four species were tested at different (w/v), which was rubbed on each plant. Biológico. Samples with absorbance dilutions, in two systemic pathosystems The plants were kept in a greenhouse reading values three times higher than involving zucchini (C. pepo ‘Caserta’) and visually inspected for 15 days those of negative controls (healthy and the CMV and ZYMV viral isolates. to observe the induction or not of samples) were considered positive. Extracts prepared with P. ambigua symptoms and determine percentage and S. langsdorffii were the most Statistical analyses inhibition, calculated as explained in efficient in the two pathosystems. InC. Statistical tests using a completely the previous item. Leaf samples were pepo ‘Caserta’ / ZYMV, viral infection randomized design were conducted collected for subsequent serological percentage inhibition greater than or to compare the absorbance values of testing. equal to 70% was observed up to a an ELISA assay of C. pepo ‘Caserta’ 1:640 dilution (Figure 2A). Similar Assessment of maintenance of leaf samples treated with extracts from inhibitions were obtained by Tozetto the inhibitory properties of leaf different Brazilian native species at et al. (2002). In the case of the C. pepo extracts at different temperatures and different dilutions and periods, 15 days ‘Caserta’ / CMV pathosystem, the preparation methods after CMV and ZYMV inoculation. same efficiency as P. ambigua and S. Pisonia ambigua and S. langsdorffii Sampling involved 10 replicates per langsdorffii LEs was detected only at LEs, the most efficient in all treatments treatment. The absorbance values of 1:40 dilution (Figure 2B). In contrast in both pathosystems, were selected to the uninfected plants (negative results) to that observed with ZYMV infection, evaluate a number of properties, such were used in statistical analyses and the LE of G. opposita was efficient in as the maintenance of antiviral activity treatments, where fewer than three inhibiting CMV infection in C. pepo when prepared using different methods uninfected plants were not considered. in 70% of the plants, but only at 1:40 and stored at different temperatures. Multiple comparisons were performed dilution. Awasthi et al. (2015) reported Thus, P. ambigua and S. langsdorffii by Welch’s Analysis of Variance that ten-fold dilutions of the extracts leaves were stored at room temperature (ANOVA) followed by the Games- dramatically reduced the inhibitory away from light and humidity for around Howell test. P-values below 0.05 were activity of viral infection. The results 3 months. After drying, aqueous extracts considered significant. All statistical found in the present study contradict were prepared at a concentration of analyses were carried out using R this finding. The distinct efficiency of 1:40 (w/v), diluted at 1:80 and 1:160 software (version 4.0.0) and bar graphs G. opposita LE in inhibiting CMV and and tested in the C. pepo / ZYMV were constructed using Excel. ZYMV infections in C. pepo reinforced pathosystem. In addition, we tested the hypothesis that the inhibitory effect fresh leaf extracts at a concentration of any LEs is highly dependent on the of 1:40 (w/v), kept in sealed flasks at RESULTS AND DISCUSSION virus/host interaction. The LE of G. room temperature away from light and integrifolia was the less efficient in the humidity for 7 days. LEs stored at -20oC The half leaf experiment in the N. two pathosystems, reaching a maximum for 3 years were also tested. Six plants glutinosa / TMV pathosystem revealed percentage inhibition of 10% (Figure per treatment and six controls were used. the high inhibitory power of the extracts 2A). The plants were kept in a greenhouse and symptoms assessed for 15 days to determine the percentage inhibition. Table 1. Percentage inhibition (PI) of local lesions induced in the tobacco mosaic virus (TMV)/Nicotiana glutinosa pathosystem applying aqueous leaf extracts from native species. At the end, leaf samples from all São Paulo, IB, 2019. plants were collected for subsequent Average no. of lesions serological detection. Species/Family PI (%) Serological detection Control Treatment Viral infection was confirmed by the Guapira opposita/Nyctaginaceae 36 1.7 95.3 enzyme-linked immunosorbent assay Pisonia ambigua/Nyctaginaceae 11.5 3.2 72.0 (ELISA), using the specific panel of Gallesia integrifolia/Phytolaccaceae 19.2 4.9 74.5 polyclonal antiserum against the coat protein of ZYMV and CMV. In all Seguieria langsdorffii/Phytolaccaceae 34.4 4.6 86.6 Horticultura Brasileira 39 (2) April - June, 2021 149 MVM Pereira et al.

The percentage inhibition results amounts of antigen. The absorbance also showed lower absorbance values of CMV and ZYMV were reinforced readings of the viral load in plants were for pokeweed (P. americana treated N. when compared to those obtained by significantly lower in the samples treated tabacum samples when compared to DAS-ELISA (Figures 2C and D), which with LEs when compared to controls. PVY-infected control plants, indicating is highly sensitive in detecting minimal Serological assay (DAS-ELISA) results lower levels of PVY infections in the former (Alishiri & Rakhshandehroo, 2015). The results of ZYMV inhibition by extracts of the four species revealed that S. langsdorffii was more efficient in inhibiting viral infection, since only individuals treated with 1:640 and 1:1,280 diluted extracts were found to be infected. The LEs at 1:20 dilution of S. langsdorffii and P. ambigua were assessed for possible induced resistance in the two pathosystems, while the LE of G. opposita was evaluated only for the CMV / C. pepo interaction. Since no inhibitory effect on viral infection was observed with G. integrifolia LE, it was not evaluated as a resistance inducer in C. pepo plants. For the ZYMV / C. pepo pathosystem, the LEs of S. langsdorffii and P. ambigua obtained similar results at time intervals Figure 1. Branches of Caryophyllales species endemic to the Atlantic Forest selected to obtain of 24 and 48 h, with inhibitions greater an aqueous solution for viral inhibition tests. (A) Guapira opposita and (B) Pisonia ambigua than or equal to 80%. However, when (Nyctaginaceae), (C) Gallesia integrifolia, and (D) Seguieria langsdorffii(Phytolaccaceae). applied 72 or 96 h before inoculation, Ruler = 10 cm. São Paulo, IB, 2019. inhibition was less than 30% (Figure

Figure 2. Percentage inhibition (PI) of leaf extracts from Gallesia integrifolia, Guapira opposita, Pisonia ambigua and Seguieria langsdorffii in different dilutions against systemic viral infection: (A) zucchini yellow mosaic virus (ZYMV) / Cucurbita pepo ‘Caserta’ pathosystem, (B) cucumber mosaic virus (CMV) / C. pepo ‘Caserta’ pathosystem. Absorbance readings values at 405 nm, after serological tests of C. pepo samples treated with leaf extracts from native species, in different dilutions and inoculated with(C) ZYMV and (D) CMV. Statistical analysis was performed only for treatments that presented at least three non-infected individuals compared to those of the infected individuals of the control. For multiple comparison, Anova-Welch followed by Games-Howell test was applied. The asterisks indicate significant differences. São Paulo, IB, 2019.

150 Horticultura Brasileira 39 (2) April - June, 2021 Endemic Atlantic Forest species of Caryophyllales as inhibitors of viral infection in zucchini

3A). A similar result was reported by to that observed for the ZYMV / C. Comparison of this result with LE Elbeshehy (2017) with watermelon pepo pathosystem. The LE from G. application 24 and 48 h before ZYMV (Citrullus lanatus, Cucurbitaceae) / opposita exhibited significant results inoculation (Figure 3C) demonstrated ZYMV pathosystem. On the other hand, in the CMV / C. pepo pathosystem, a more pronounced difference, which M. jalapa LE was more efficient against similar to that obtained with LE from was confirmed by the lower p-values ZYMV inoculation in C. pepo ‘Caserta’ S. langsdorffii when applied 24, 48 and (≤0.0001). The comparisons between (Tozetto et al., 2002). 72 h before viral inoculation. In the 96-h different times only detected a difference In addition, as observed in the pre-treatment, the percentage inhibition between the 24 and 72 h values of leaves test to determine maximum dilution, obtained with this extract (41%) was pre-treated with S. langsdorffii LEs; significantly lower absorbance readings higher than that of the two other species however, the p-value (p = 0.044) was were found in samples with high (Figure 3B). close to the 0.05 value that is usually percentage inhibition (24 and 48 h) Although the multiple comparison adopted in statistical analyses as the for the ZYMV / C. pepo pathosystem performed between absorbance values threshold for a significant difference. when compared to controls (Figure 3C), from zucchini leaves pre-treated with The decline in the number of infected corroborating viral infection inhibition. LEs of S. langsdorffii, P. ambigua and plants in the two pathosystems (ZYMV Similar to the ZYMV / C. pepo G. opposita and non-treated leaves / C. pepo and CMV / C. pepo) after pathosystem, S. langsdorffii extract was (control) 48 (24, 48 and 72 h) 72 h pre-treatment with the LEs from these very efficient in inducing resistance to before CMV inoculation detected native Caryophyllales species, indicates CMV, with 85, 70 and 57% inhibition differences (Figure 3D), the Games- possible induction of the plant defense at time intervals of 24, 48 and 72 h, Howell post-hoc test was not robust system, increasing the resistance of respectively (Figure 3B). High inhibitory enough to detect where these differences these plants to a pathogen. Elbeshehy efficiency with longer time intervals occurred. The p-values between the (2017) previously suggested that LEs was observed with M. jalapa extract LEs from the three species and the from B. spectabilis must perform in the Solanum melongena (eggplant) controls were marginal (0.07≤p≤1.00), an essential function in the acquired / CMV pathosystem (Bharathi, 1999). but higher compared with p-values systemic resistance mechanism, with In the case of P. ambigua LE, the obtained from the comparisons between likely pathogenesis-related (PR) highest percentage inhibitions were LEs. As such, the differences detected protein production. Analyses are being observed in samples pre-treated 24 and by Welch’s ANOVA were more likely conducted to determine whether PR 48 h before viral inoculation, similar to occur between LEs and the controls. protein production occurs in plants pre-

Figure 3. Percentage inhibition of leaf extracts from native species sprayed at different times before inoculation with (A) zucchini yellow mosaic virus (ZYMV) and (B) cucumber mosaic virus (CMV) in Cucurbita pepo ‘Caserta’. Average absorbance readings at 405 nm, after serological tests of C. pepo samples treated with leaf extracts from P. ambigua and S. langsdorffii, at different times and inoculated with(C) ZYMV and (D) CMV. Leaf extract of G. opposita was also applied at different times before inoculation with CMV (B, D). The asterisks indicate significant differences.São Paulo, IB, 2019.

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Figure 4. (A) Percentage inhibition of leaf extracts from native species sprayed 30 minutes before inoculation with zucchini yellow mosaic virus (ZYMV) in Cucurbita pepo ‘Caserta’. Extracts prepared with dry leaves and applied in different dilutions. (B) Average absorbance readings at 405 nm, after serological tests of C. pepo samples treated with leaf extracts from P. ambigua and S. langsdorffii. Different letters over each bar correspond to significant differences at 0.05%, according to Games-Howell test. São Paulo, IB, 2019.

Figure 5. (A) Percentage inhibition of leaf extracts from native species stored for 3 years at -20°C and applied 30 minutes before inoculation with zucchini yellow mosaic virus (ZYMV) in Cucurbita pepo ‘Caserta’. Extracts prepared with fresh leaves. (B) Average absorbance readings at 405 nm, after serological tests of C. pepo samples treated with leaf extracts from P. ambigua and S. langsdorffii. Different letters over each bar correspond to significant differences at 0.05%, according to Games-Howell test. São Paulo, IB, 2019. treated with extracts from native species. the same as that observed for fresh leaf recommended to zucchini producers Surveys in Brazil have shown that extract (Figure 2A), it was not detected as a management strategy for disease ZYMV occurrence is uninterrupted and for P. ambigua. Rasoulpour et al. (2018) caused by ZYMV. prevalent in zucchini (Lima & Alves, also found that the inhibitory effect With respect to storage tests, the 2011). Thus, stability and storage tests of the aqueous extract obtained from inhibitory effect of ZYMV infection of were conducted to provide information the dry clades of Opuntia ficus-indica zucchini was maintained for at least 3 to zucchini producers on obtaining and (Cactaceae, Caryophyllales) against years in extracts stored at -20°C (Figure storing leaf extracts. The LEs prepared CMV infection in the fava bean was 5). The efficiency of S. langsdorffii from dry leaves of P. ambigua and S. maintained. LE was as good as that of fresh crude langsdorffii, at 1:40, 1:80 and 1:160 Similar to fresh leaf extracts, there extract (Figure 2A), but a decrease dilutions (w/v) were evaluated in the was a reduction in viral particles in in virus-infection inhibition of more ZYMV / C. pepo pathosystem. The LE samples with great inhibitory effect than 50% was detected with stored from S. langsdorffiiexhibited inhibitory using dry leaf extracts. No significant P. ambigua LE. On the other hand, activity in the three dilutions (90, 80 and difference between the 1:40 and 1:80 S. langsdorffii LE kept for 7 days at 70%, respectively), while the LE from dilutions of P. ambigua LEs was room temperature inhibited ZYMV P. ambigua induced 70% inhibition at a observed, and the 1:80 dilution of S. infection in C. pepo only up to 40%, concentration of 1:40 and lost inhibition langsdorffii LE was lower than 1:40 demonstrating a significantly lower capacity as dilution increased (Figure (Figure 4B). Thus, extracts prepared viral load than that of controls (data not 4A). While the inhibition performance from the dry leaves of these two species shown). P. ambigua LE kept at room of dry leaf extract of S. langsdorffii was at a dilution of up to 1:80 can be temperature oxidized, precluding this

152 Horticultura Brasileira 39 (2) April - June, 2021 Endemic Atlantic Forest species of Caryophyllales as inhibitors of viral infection in zucchini test. Comparison of the absorbance exhibit excellent potential as resistance complex interactions. Annual Review of values from non-infected samples of inducers and deserve more in-depth field Entomology 63: 169-91. ELBESHEHY, EKF. 2017. Inhibitor activity of plants treated with LEs revealed that study, in order to assess the viability different medicinal plants extracts fromThuja plants treated with P. ambigua LEs of their application in minimizing the orientalis, Nigella sativa L., Azadirachta obtained higher values than those treated damage caused by plant viruses. indica and Bougainvillea spectabilis against with their S. langsdorffii counterparts. zucchini yellow mosaic virus (ZYMV) infecting Citrullus lanatus. Biotechnology & However, both LEs showed lower ACKNOWLEDGEMENTS Biotechnological Equipment 31: 270-279. doi: absorbance values when compared to 10.1080/13102818.2017.1279572. the controls (Figure 5B), reinforcing The Research Support Foundation GRASSO, S; SHEPHERD, RJ. 1978. Isolation and partial characterization of virus inhibitors virus inhibition. of São Paulo State (FAPESP - proc. According to Awasthi et al. (2015), from plant species taxonomically related to 2016/25708-4), and the National Phytolacca. Phytopathology 68: 199-205. inhibitory activity was lost after one Council for Scientific and Technological HERNÁNDEZ-LEDESMA, P; BERENDSOHN, week storage for several of the extracts Development (CNPq) for the funding WG; BORSCH, T; MERING, SV; AKHANI, tested. However, M. jalapa LE remained and scholarships granted. H; ARIAS, S; CASTAÑEDA-NOA, I; EGGLI, active for up to one month, reaching U; ERIKSSON, R; FLORES-OLVERA, H; FUENTES-BAZÁN, S; KADEREIT, G; percentage inhibition of eggplant REFERENCES KLAK, C; KOROTKOVA, N; NYFFELER, infection by CMV of 100, 88, 75, 50, R; OCAMPO, G; OCHOTERENA, H; 44 and 0% when stored for 4, 8, 12, 16, OXELMAN, B; RABELER, RK; SANCHEZ, ALISHIRI, A; RAKHSHANDEHROO, F. 2015. 20 and 24 days at room temperature, A; SCHLUMPBERGER, BO; UOTILA, P. Study on the antiviral activity of aqueous 2015. A taxonomic backbone for the global respectively (Bharathi, 1999). pokeweed (Phytolacca americana L.) extracts synthesis of species diversity in the angiosperm Our results indicate that three of the against Potato virus Y. Iranian Journal of order Caryophyllales. Willdenowia 45: 281- four native species exhibited excellent Medicinal and Aromatic Plants 31: 479-487. 383. doi: 10.3372/wi.45.45301. viral infection inhibitory potential for APG IV. 2016. 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