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The ornamental plant industry is growing every year (Silva et al., 2018), although the environmental and technological influences of intensive production make the production of diseases-free plants difficult. Due to this, establishing efficient strategies to prevent and manage ornamental plant diseases is imperative (Barrera-Necha and Bautista-Baños, 2016). The chrysanthemum (Chrysanthemum morifolium) is one of the most important ornamental crops in the production of flowers globally (Anderson, 2007; Kumar et al., 2011; Olejnik et al., 2021). This plant is originally from eastern Asia and it adapts to diverse climates (Villanueva-Couoh et al., 2005). Its surface registered in Mexico was 3,203 ha, with a production worth 1,915,691 pesos (SIAP, 2020).
The chrysanthemum is susceptible to several pathogens, particularly when grown on a large scale. The leaf blight caused by Alternaria spp. is one of the main diseases that affect its production (Xu et al., 2010). Several Alternaria species are responsible for this disease, including Alternaria alternata, A. tenuissima and A. chrysanthemi, which can infect plants individually or in interactions, reducing its quality and value in the market (Domínguez-Serrano et al., 2016; Trolinger et al., 2018; Xu et al., 2010). Symptoms appear on the edges of the leaves with yellow, maroon to blackish lesions and the presence, in some cases, of chlorotic halos. The progress of the disease is visible with the necrotic areas of variable sizes that can cause the defoliation of the plant. When the disease is severe, the flower petals display reddish-maroon lesions, which then become completely necrotic (Borah et al., 2019; Domínguez-Serrano et al., 2016; Trolinger et al., 2018). Without an adequate control, it can lead to the death of the plant (Deng et al., 2012; Xu et al., 2010; Zhu et al., 2014).
The main control strategy against leaf blight is using synthetic fungicides (Kumar et al., 2017; Trolinger et al., 2018; Villanueva-Couoh et al., 2005; Xu et al., 2010). However, the recurring and inadequate use of these products has caused problems of resistance in fungal strains, environmental pollution and intoxications in producers (Carvalho, 2017; Kim et al., 2017; Barrera-Necha y Bautista-Baños, 2016; Shuping and Eloff, 2017). This situation demands an exploration of biorational alternatives to control the disease. Among these are natural compounds such as chitosan, essential oils and plant extracts (Barrera-Necha and Bautista-Baños, 2016). The latter have proven to have the potential to control phytopathogenic fungi due to their high effectiveness, low cost and low toxicity on beneficial organisms (Breda et al., 2016; Ngegba et al., 2018; Tomazoni et al., 2017). However, there are few studies on the use of plant extracts to control diseases in ornamental plants (Barrera-Necha and Bautista-Baños, 2016). Under in vitro conditions, Nivedha et al. (2019) evaluated 24 plant extracts for the control of A. alternata in Jasminum grandiflorum. The Allium sativum, Datura metel and Azadirachta indica extracts presented the highest fungicidal activities, with inhibitions in the fungal mycelial growth of 100, 68 and 59%, respectively. There are also reports of Capsicum annuum, Helianthus annuus and Tagetes erecta aqueous extracts (in concentrations of 4-8%), effective against Fusarium oxysporum f. sp. gladioli, with mycelial inhibition ranges of 45-57%, 33-85 and 54-79%, respectively (Riaz et al., 2008).
Acalypha gaumeri and Bonellia flammea are native plants of the Yucatan Peninsula that have displayed antifungal effects against A. alternata, A. chrysanthemi, Curvularia verruculosa, C. lunata, Corynespora cassiicola, Bipolaris setariae, Colletotrichum gloeosporioides, Exserohilum rostratum, F. oxysporum, Helminthosporium sp. and Lasiodiplodia parva (García-Sosa et al., 2011; Gamboa-Angulo et al., 2008; Moo-Koh et al., 2014; Vargas-Díaz et al., 2014). In a previous in vitro study, the aqueous extracts of the root of A. gaumeri and the cortex of B. flammea had an inhibiting effect on mycelial growth (ICM) and sporulation (IS) of A. chrysanthemi (ICM = 51 - 57%, IS = 77 - 82%, respectively) (Vargas-Díaz et al., 2014).
The inhibition of phytopathogenic fungi using plant extracts is well documented at a laboratory level, although the studies to determine their efficacy in the field are scarce (Castillo-Reyes et al., 2018). On the other hand, aqueous extracts have been documented to be safer and easier to use in the field than other extraction methods using solvents. Despite the species of A. gaumeri and B. flammea presenting inhibiting effects against phytopathogenic fungi, they lack field studies to control diseases in plants. Based on this, the aim of this study was to identify the causal agent of leaf blight in chrysanthemum and to control it in the field using Acalypha gaumeri and Bonellia flammea aqueous extracts.
Materials and methods
Isolation and characterization of the phytopathogen that causes leaf blight. Alternaria sp. was isolated from chrysanthemum leaves and stems with signs and symptoms related to foliar blight. The samples were collected in the “Nicte-Ha” Floricultural Unit, in the municipal area of Chocholá, Yucatan, Mexico (20°41’ N and 89°49’ W). Cuts sized approximately 1 cm2 were made on the infected material, which were then disinfested in a 2% sodium hypochlorite solution for 2 min. Four to five pieces of plant tissue were placed in Petri dishes with potato-dextrose-agar medium (PDA) and incubated at 28 ºC (Villanueva-Couoh et al., 2005). After three days, the fungus was purified using monosporic cultures. To achieve the sporulation of the fungus, the malt extract agar medium was used, and from these, the fungus was identified by morpho-taxonomic characteristics that included color and type of mycelium, number of conidia and conidiophore (Barnett and Hunter, 1972; Simmons, 2007), measurement of the length and width of 100 conidia using a compound microscope (Standard Model K7) and at a 400x magnification.
Likewise, it was corroborated with the molecular identification of the phytopathogen. For this purpose, the fungal strain was registered as CICY004 and it was cultivated in PDA medium in Petri dishes and incubated at 25 °C and 12 h of light/ 12 h of darkness for four days. The genomic DNA was extracted following the procedure by Johanson and Jeger (1993). The reaction mixture (1X) contained 100 ng of gDNA, 2 mM MgCl2, 200 mM dNTPS, 0.4 mM of every first ITS1 and TS4 (White et al., 1990), and 1U of Taq polymerase, in a total volume of 25 mL. The PCR reaction was carried out with an initial denaturalization of 95 oC for 3 min and 30 cycles with 1 min at 95 oC, 45 s at 53 oC and 30 s at 72 oC. After the cycles, an elongation phase took place, lasting 3 min at 72 oC.
The PCR products were analyzed by electrophoresis in agarose gel at 1% (Invitrogen®). The amplified products were purified with a PCR purification kit (Gene Clean II, Roche) and were later sequenced in the Sequencing Center in Davis, CA (http://www.davissequencing.com) using 100 ng of the PCR product. Both DNA stands were sequenced, using oligo ITS1 for the sense strand and oligo ITS4 for the antisense strand.
The sequences obtained were edited using the program BioEdit Sequence Alignment (Hall, 1999) to eliminate the sequences corresponding to the primers and the inadequately sequenced regions (noise with Ns). Once edited, the antisense sequence became a sense sequence with the use of the Reverse Complement tool (https://www.bioinformatics.org/sms/rev_comp.html). In both sequences, they were aligned using the program ClustalW (Madeira et al., 2019) to obtain a consensus sequence in which each base was confirmed by both strands; a consensus sequences with 533 nucleotides was obtained, and it was compared with the database of the NCBI (https://www.ncbi.nlm.nih.gov/) and the RDP “Ribosomal Database Project” (https://rdp.cme.msu.edu/classifier/classifier.jsp) using the BLAST algorithm. The 533-nucleotide sequence was deposited in the GenBank with accession number MH846127.
A BLASTN analysis was carried out with sequence MH846127 and one accession of each species was chosen among the first 100 comparisons that were homologous with sequence MH846127. Additionally, accession MN153803 from strain HN4-9 ofA. eichhorniae was included, since it was not among the first 100 counterparts. The analysis was carried out with the program MEGA X using the Maximum Likelihood method (Tamura and Nei, 1993).
The pathogenicity of strain CICY004 was determined in 15 healthy chrysanthemum plants under protected conditions. The plants were superficially cut in the axial part of the leaves with a paintbrush and 20 µL of a suspension of 2.5 × 106 spores mL-1 were inoculated. The plants were placed in separate plastic containers, which were covered to regulate the temperature and moisture for 2 days. After five days, the pathogen was reisolated from the necrotic lesions on the leaves (Riego et al., 1997).
Preparation of the aqueous extracts. The aqueous extract of the dehydrated plant material of A. gaumeri roots and the cortex of B. flammea (30 g of each) was obtained by infusion (20 min) in 500 mL of distilled water at boiling point (Herrera-Parra et al., 2009). The infusion was filtered through paper and diluted up to 1000 mL to obtain the aqueous extract at 3% (30 g L-1, p/v) (Vargas-Díaz et al., 2014). The extract was stored at room temperature (less than 24 h) until its application on the field.
Evaluation of extracts in the control of the disease. To ensure the presence of the pathogen in the crop in the field, strain CICY004 was grown for 15 days in PDA medium at 23 °C. From this crop, a suspension of 2.5 × 106 spores mL-1 was made (Vargas-Díaz et al., 2014). The crop was established in the months between March and May, in the Unit Limones Cheé in the Ulu´umil lool floricultural area, in the municipal area of Maní, Yucatan, Mexico (20°39’ N and 89°40’ W). C. morifolium cv. Polaris Yellow trimmings, aged 20 days, were used. These were provided by the “Nicte-Ha” Floricultural Unit in the municipal area of Chocholá, Yucatan, Mexico. The trimmings were transplanted in beds, 1m wide × 12m long, and a local mixture of soil, organic bovine manure and gravel was used as a substrate in a 2:2:1 proportion. The experimental design was a completely randomized block design, with four repetitions per treatment; for each repetition, plots measuring 1m × 2.5 m were used. When the plants reached a height of 30 cm, each experimental plot was inoculated with a suspension of 2.5 × 106 spores mL-1 of the fungal strain indicated. After eight days of inoculation, the plants were sprayed on a weekly basis with the treatments for seven weeks. The evaluated treatments were T1: aqueous extract of the cortex of B. flammea (30 g L-1), T2: aqueous extract of the root of A. gaumeri (30 g L-1), alongside two controls, T3: negative control (water) and T4: Captan commercial fungicide (2 g L-1).
The disease was controlled by estimating its severity, with the use of a diagramatic logarithmic scale of severity for the C. morifolium-A. chrysanthemi pathosystem (Villanueva-Couoh et al., 2005). Measurements began when observing the symptoms of the disease (eight days after inoculation with the phytopathogen) and continued every week with random sampling, and in each repetition, a total of 20 central plants, out of which 30 base leaves of each plant were considered. Based on the percentage of severity, disease progress curves were built and the Area Under the Disease Progress Curve (AUDPC) was estimated using the trapezoidal integration method (Campbell and Madden, 1990), apparent infection rates with Weibull’s description model, with the reverse parameter b -1 (Thal et al., 1984) and the final severity of the disease (Yfinal) with the final evaluation, and which was used to calculate the effectiveness of the control of the disease (Abbott, 1925).
In addition, as growth variables, the number of buttons and the diameter of a bunch were recorded in 20 central plants. The experiment was carried out twice more and the results were averaged for their statistical analyses. The general management of the crop was carried out according to the traditional practices of the farmer. At the end of the experiment, the fungus was isolated to verify the presence of strain CICY004.
Data analysis. The data obtained from the epidemiological parameters, as well as the growth variables, were analyzed using the SAS for Windows version 9 computer package using a one-way analysis of variance and a test of comparison of averages (Tukey, p≤0.01).
Results
Morphological and molecular identification. The strain of fungus CICY004 in a malt extract medium displayed a velvet-like growth, initially olive-colored on the edges, and later brown. On the back of the Petri dish observed in black color. The conidia displayed an obclavate or cylindrical shape, with a dark color. In its morphometry, it displays a length of 52 to 82 μm by 12.5 to 15 μm in width, at its widest, with 5 to 9 transversal septa and 0 to 4 longitudinal ones, frequently in chains of twos or on their own (Figure 1). The morphological analysis of strain CICY004 displayed differences with the strains reported by Domínguez-Serrano et al. (2016), since it presented larger conidia (52-82 × 12.5-15 µm) than A. alternata (15-35 × 7.5-12.5 µm) and then A. tenuissima (30-67,5 × 7,5-12,5 µm). Likewise, strain CICY004 presented short chains with one or two conidia, whereas in A. alternata, chains presented 10-37 conidia, and in A. tenuissima, 5-12 conidia (Barnett and Hunter, 1972; Simmons, 2007). These data confirmed that strain CICY004 is different, and according to the description of the fungus, it corresponded to the species A. chrysanthemi (Barnett and Hunter, 1972; Simmons, 2007). The pathogenicity of the strain was proven after being inoculated and reisolated in chrysanthemum plants.
Figure 1 Morphological characteristics of strain CICY004 (A. chrysanthemi), A). Morphology of the culture in malt extract agar medium, B) conidia of strain CICY004.
In the BLAST analysis of the ITS region of strain CICY004, it was homologous to sequences that correspond with Alternaria with a 100% coverage and a 99.81% identity, therefore confirming that it belongs to this genus. To confirm that the strain registered as CICY004 corresponds with some of the species of Alternaria previously reported in Mexico (Domínguez-Serrano et al., 2016), the sequence of the ITS region of CICY004 was compared with those strains (A. alternata KF728748, KF728750 and A. tenuissima KF728749, KF728751), and an identity of 99.62% was found with both species, discarding that CICY004 is one of them. Based on morphological identification and sequence analysis, the strain with record CICY004, was identified as A. chrysanthemi. This sequence was deposited in the GenBank as A. chrysanthemi CICY004 with accession number MH846127.
Effect of extracts in the field over the control of the disease. The symptoms of leaf blight disease in the crop were presented 37 days after transplanting (dat). In the negative control (water), the progress of the disease was higher than in the rest of the treatments (Figure 2). Based on the AUDPC and Yfinal, the aqueous extracts of A. gaumeri (T2) (165% a day, 8 and 67%, respectively) and cortex of B. flammea (T1) (186% a day, 13 and 50%, respectively) reduced the disease severity, with a significant difference with the negative control (T3) (369% a day, 25 and 0%, respectively). Likewise, the application of these extracts was significantly equal to the fungicide Captan® (T4) (179% a day, 14 and 45%, respectively) (Tables 1 and 2). However, when the speed of the diseases was estimated using the infection rate with the Weibull description model (b -1), only the aqueous extract from A. gaumeri T2 (0.017 unit day-1) significantly reduced the progress of the disease (Table 1) (Figure 3).
Figure 2 Progress curves of the severity of leaf blight caused by A. chrysanthemi on the chrysanthemum crop and effect of the application of treatments T1: Aqueous extract of the cortex of B. flammea, T2: Aqueous extract of the root of A. gaumeri, T3: Negative control (water) and T4: Captan ® fungicide.
Table 1 Effect of the application of treatments on the epidemiological intensity of leaf blight caused by A. chrysanthemi on the chrysanthemum crop.
| Tratamientos | ABCPE(% día-1) | Tasa de infecciónaparente (Weibull 1/b % día-1) | Ajuste del modeloWeibull (r2) | Yfinal(%) |
|---|---|---|---|---|
| T1: Extracto acuoso de cortezade B. flammea | 186 b | 0.022 a | 0.97 | 13 b |
| T2: Extracto acuoso de raíz de A. gaumeri | 165 b | 0.017 b | 0.98 | 8 b |
| T3: Control negativo (agua) | 369 a | 0.025 a | 0.96 | 25 a |
| T4: Fungicida Captan®(2 g L-1) | 179 b | 0.023 a | 0.96 | 14 b |
| DE | 12 | 0.003 | - | 1.3 |
Means with the same letter between columns are statistically equal (Tukey, p≤0.05). DS: Standard Deviation.
Figure 3 Effect of treatments on the control of leaf blight in chrysanthemum, T1: Aqueous extract of the cortex of B. flammea, T2: Aqueous extract of the root of A. gaumeri, T3: Negative control (water) and T4: Captan ® fungicide.
In the chrysanthemum crop growth variables: number of buds and bunch diameter, the treatments that included the extracts and the synthetic fungicide significantly improved these variables (p≤0.01) (Table 2).
Table 2 Effectiveness of the aqueous extracts of plants to control leaf blight and growth variables in chrysanthemum.
| Tratamientos | Efectividad (%) | Variables Agronómicas | |
|---|---|---|---|
| Número de Botones (n planta-1) | Diámetro de racimo (cm) | ||
| T1: Extracto acuoso de cortezade B. flammea | 50 az | 18 a | 15 a |
| T2: Extracto acuoso de raíz de A. gaumeri | 67 a | 17 a | 14 a |
| T3: Control negativo (agua) | 0 b | 12 b | 10 b |
| T4: Fungicida Captan®(2 g L-1) | 45 a | 19 2 a | 14 a |
| DEy | 5.57 | 2.63 | 1.44 |
yDE: standard deviation of the mean. zMeans with the same letter between columns are statistically equal (Tukey, p≤0.05).
Discussion
The diseases caused by fungi severely affect the chrysanthemum industry (Borah et al., 2019; Trolinger et al., 2018). This study identified A. chrysanthemi as the causal agent of leaf blight in the chrysanthemum in Mexico. This species has been reported in chrysanthemum in other countries such as Australia, the United States, the Netherlands and India (Sobers, 1965). In Mexico, Domínguez-Serrano et al. (2016) reported the species of A. alternata (KF728748, KF728750) and A. tenuissima (KF728749, KF728751) as causal agents of blight in chrysanthemum, the morphological and molecular analyses of which do not coincide with the isolated strain in this study. The alignment with the previously reported sequences of A. alternata (KF728748, KF728750) and A. tenuissima (KF728749, KF728751) present two and three different nucleotides with the strain CICY004. It is important to mention that Domínguez-Serrano et al. (2016) found only one difference in the region they studied (540 nucleotides) of the ITS of A. alternata and A. tenuissima; only one insertion of a “T” nucleotide in the sequence of the second and absent in A. alternata, whereas the rest was identical. This difference was accepted as a criterion to distinguish both species, since the authors sequenced each species 5 times and that difference was reproducible.
In this investigation, the differences found between CICY004, A. alternata and A. tenuissima were also few and accepted, since the sequence of CICY004 corresponds to a consensus sequence, 100% confirmed with the sense and antisense strand sequences. These nucleotide differences are informative and tell CICY004 apart from A. alternata and A. tenuissima, as well as from A. eichhorniae. The morphology revealed that it is A. chrysanthemi, making this the first report of the ITS sequence of this phytopathogen, since it did not exist earlier in the GenBank or in the RDP. This study is now a reference for future investigations on A. chrysanthemi as the causal agent of leaf blight in chrysanthemum.
In the control of leaf blight, both A. gaumeri and B. flammea aqueous extracts and the Captan® fungicide displayed the same effect in stopping the progress of the disease and improved upon what was reported by Kumar et al. (2017) when evaluating garlic extracts (effectiveness of 36.64%) in the same crop. In addition, the A. gaumeri and B. flammea aqueous extracts displayed a greater effect in comparison with the Eucalyptus globolus and Ocimum sanctum aqueous extracts at 5% against the early potato blight (20.83 and 25.37%) (Debbarma et al., 2017). These results indicated that the A. gaumeri and B. flammea extracts at 3% can reduce the severity caused by A. chrysanthemi, and the information previously reported on the in vitro tests against this pathogen (Vargas-Díaz et al., 2014) was confirmed.
In parameters AUDPC and Yfinal, both extracts turned out to be as effective in the reduction of the severity as the Captan® fungicide. A study by Ortega-Centeno et al. (2010) reported in cauliflower, broccoli and radishes (Brassicaceae), the same effectiveness of control as with synthetic fungicides (captan, amistar and manzate) against Uromyces transversalis, which causes rust in gladiolus rust.
The plants A. gaumeri and B. flammea are considered endemic to the Yucatán Peninsula, meaning there is scarce information on their phytochemical and biological properties (Hernández-Bolio et al., 2019). A. gaumeri is a plant on which there are few reports regarding its antifungal activity (Gamboa-Angulo et al., 2008; Vargas-Díaz et al., 2014). In the genus there have been chemical families found, depending on the solvent used (terpenes, steroids, tannins, saponins alkaloids, anthocyanins, polyketides and flavonoids) mainly responsible for antifungal activities (Adesina et al., 2000; Gutierrez-Lugo et al., 2002; Hungeling et al., 2009; Seebaluck et al., 2015).
Regarding B. flammea, from methanolic root extracts, García-Sosa et al. (2011) reported an inhibiting effect against C. gloeosporioides. Sánchez-Medina et al. (2010) identified the presence of a sakurososaponin from the aqueous residue from the liquid-liquid partition of the extract, with a cytotoxic activity and against C. gloeosporioides. Sakurososaponin is one of the main metabolites responsible for the antifungal activity of B. flammea, and it is known to have the ability to form complexes with sterols of the cell membrane, which causes a loss in its integrity and the death of the fungus (Morrissey and Osbourn, 1999).
When growing ornamental plants, the esthetic damage on the foliage and flowers caused by phytopathogens is considered crucially important, since it reduces their quality, shelf life and price for sale (Barrera-Necha and Bautista-Baños, 2016). The reduction in the intensity of the disease with the application of extracts and the fungicides allowed for a higher number of buttons and diameter of the bunches, with a significant difference with the negative control, which is crucial to obtain higher yields in the production of chrysanthemum (Kumar et al., 2011).
In addition to being easily obtainable for application on the field by farmers, aqueous extracts are environmentally safer in comparison with other methods of extraction with solvents. This represents an important step in the development of environmentally friendly plant-based pesticides. This study is the beginning of future investigations that contributes to proposing strategies for the control of diseases in the sustainable planting of chrysanthemum.
