In Vitro Antifungal Activity of Plant Extracts on Pathogenic Fungi of Blueberry (Vaccinium sp.)

Three pathogenic fungi of blueberry (Vaccinium spp.) responsible for dieback disease, identified as Pestalotiopsis clavispora, Colletotrichum gloeosporioides and Lasiodiplodia pseudotheobromae, were isolated in the northwestern region of the state of Michoacán, Mexico. The mycelial growth in vitro of these fungi was inhibited by extracts from Lantana hirta, Argemone ochroleuca and Adenophyllum porophyllum, medicinal plants collected in Sahuayo, Michoacán, Mexico. The extracts showed different degrees of inhibition; the most effective were: M5L extract from L. hirta and M6LFr extract from A. ochroleuca, both of which inhibited 100% of the mycelial growth of P. clavispora and C. gloeosporioides; and M4LS extract from A. porophyllum, which inhibited 100% of the mycelial growth of the three pathogens. The extracts were fractionated by thin layer and column chromatography, and the most active fractions were analyzed by gas chromatography-mass spectrometry. The major compounds identified in L. hirta extract were Phytol and α-Sitosterol. The compounds identified in A. ochroleuca were Toluene and Benzene, 1,3-bis(3-phenoxyphenoxy)-. In A. porophyllum, the compound identified was Hexanedioic acid, bis(2-ethylhexyl) ester. These results show the potential of L. hirta, A. ochroleuca and A. porophyllum as a source of antifungal compounds.


Introduction
The main producers of blueberry are the United States, Canada and Poland; Mexico is ranked 18th. In this country, the major producing states are Michoacán and Jalisco, with 37,090 t produced in 2019 [1]. In the northwestern region of the state of Michoacán, Mexico, the production of blueberry is affected by various factors; among the most important are diseases caused by Pestalotiopsis sp.; Colletotrichum sp.; and other fungi belonging to the family Botryosphaeriaceae, such as Lasiodiplodia theobromae, Neofusicoccum ribis and Botryosphaeria sp. [2][3][4]. These fungi have the ability to affect fruits and stems, causing the disease known as dieback [5,6], which is responsible for significant losses in the production of blueberries and the increase in production costs related to fungicides. The continuous and indiscriminate use of fungicides causes adverse effects to human health and the environment [7], which, together with the increasing demand for blueberries, current regulations on chemical pesticides and the emergence of resistant pathogens, justifies the search for new active molecules that can be used as control agents against phytopathogenic fungi [7,8] and that can be included in programs associated with the integrated management of pests.
Secondary metabolites with fungicidal properties have biological activity against phytopathogenic fungi and are, therefore, a viable control option. They are biodegradable to non-toxic products and could be a sustainable tool as bio-pesticides in integrated pest management programs [9][10][11]. The main source of these metabolites can be found in medicinal plants, of which more than 200 species with this activity have been studied and reported in Mexico [12]. The state of Michoacán, Mexico, has an enormous wealth of plant species and important cultural traditions on the use of medicinal plants, such as "Siete Colores", "Chicalote" and "Árnica de Cerro", which are widely used as analgesics, and antidiarrheal, anti-inflammatory and antimicrobial remedies [13][14][15]. To our knowledge, no studies have been conducted to identify the bioactive secondary metabolites of these species or to evaluate their antifungal activity against the major pathogenic fungi of blueberries grown in the northwestern region of the state of Michoacán, Mexico.
Thus, the objectives of this study were to identify the main fungi responsible for causing dieback disease in blueberries; to evaluate the in vitro antifungal activity of extracts from Lantana hirta, Argemone ochroleuca and Adenophyllum porophyllum against fungi associated with this disease; and to identify the bioactive secondary metabolites present in these plant species.

Isolation of Pathogenic Fungi of Blueberry
Diseased plants with symptoms of wilt, blight, canker and dieback were collected from plots in the following four municipalities that produce blueberries in the northwest of the state of Michoacán, Mexico: Emiliano Zapata, Los Palillos, La Magdalena and San Gregorio. The fungi were isolated according to the procedure previously published [16]. For this, fragments of diseased tissue of approximately 10 × 10 mm were obtained from the diseased plants collected; disinfested with 3% (v/v) sodium hypochlorite for 2 min, rinsed three times with sterile distilled water; and dried on sterile paper napkins for 20 min, in a laminar flow cabinet (CHC Biolus ® , Daejeon, Korea). Five fragments of sick tissue were transferred per Petri dishes containing potato-dextrose-agar culture medium (PDA, Bioxon ® , Estado de México, Mexico) and incubated at 28 • C for 4-7 days, until fungal growth. The fungal isolates were purified using the hyphal tip technique, as follows: the colonies were transferred to water-agar culture medium (20%) (Bioxon ® ), where they were left to grow for 3 days at 28 • C; hyphal tips were then removed and transferred individually to PDA medium. The fungi grown on PDA were kept on sterile filter paper (2 cm 2 ) for 15 days. Finally, they were placed in a sterile container and left in refrigeration at 5 • C until further use.

Molecular Identification of Fungi
The molecular identification of the fungi was carried out by polymerase chain reaction (PCR) using oligonucleotides specific for the ITS regions of rDNA. The extraction and quantification of DNA was performed according to the method of Raeder and Broda [17]. The fungal DNA was purified using The Wizard ® Genomic DNA Purification Kit (A1120, Promega Corp., Madison, WI, USA). The PCR was performed using the primers ITS1-Fwd (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4-REV (5 -TCCTCCGCTTATTATTGATATGC-3 ). PCR reactions were performed in a final volume of 25 µL using the GoTaq ® Green Master Mix kit (M7122, Promega Corp. Madison, WI, USA) 1 X; 5 µM of the primer ITS1-Fwd primer; 5 µM of ITS4-Rev; and 25 ng of genomic DNA. Amplification was performed in a C1000 thermal cycler (Bio-Rad ® , Germany) under the following conditions: initial denaturation at 94 • C for 5 min, followed by 35 cycles of denaturation at 95 • C for 30 s, 58 • C for 30 s, 72 • C for 1 min and a final extension at 72 • C for 7 min. The PCR products were separated by agarose gel electrophoresis (2%) at 80 V for 40 min, and visualized using SYBR Gold ® (Invitrogen, Carlsbad, CA, USA). DNA fragments of 700 bp were selected for purification with ExoSAP-IT ® (N/P 78200, USB Affymetrix, Inc., Cleveland, OH, USA). The purified fragments were sequenced using primer ITS1-Forward (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4-Rev (5 -TCCTCCGCTTATTATTGATATGC-3 ) with the ABI PRISM BigDye ® Terminator sequencing kit v3.1 (P/N 4336917, Applied Biosystems, Foster City, CA, USA). Fragment analysis was conducted in a Genetic Analyzer 3130 sequencer (Applied Biosystems ® , HITACHI Tokyo, Japan). The sequences were assembled using SeqMan software 8 (LaserGene) (DNASTAR ® , Madison, WI, USA) and analyzed using the GenBank database (www.ncbi.nlm.gov accessed on 8 May 2014).

Pathogenicity Tests
Pathogenicity tests were performed on months-old blueberry plants (cv. Biloxi) obtained from Centro Regional Universitario Centro Occidente (CRUCO) of Chapingo Autonomous University, Campus Morelia, Michoacán. Each plant was transplanted into plastic pots (8 L) with a soil (80%) and chicken manure vermicompost-based substrate (20%). Pure four days-old fungal cultures were used for inoculation. The experimental design consisted of randomized blocks with four treatments (three fungi and a single control with PDA only) and five repetitions. Three stems of each plant (experimental unit) were inoculated through a 1 cm incision on which a disc (5 mm in diameter) with PDA and mycelium of the corresponding strain was placed; parafilm was used to keep the disc firmly on the stem. The plants were inspected daily until the onset of symptoms.

Obtaining Crude Extracts and Fractions
The plants were sectioned into roots, stems, leaves, flowers and fruits, which were disinfected with 1% sodium hypochlorite, rinsed with distilled water and dried with absorbent paper [18]. The crude extracts were obtained from the following plant organs: leaf (L) and flower (Fl) of L. hirta; leaf-fruit (LFr) and root (R) of A. ochroleuca; and leaf-stem (LS) and leaf of A. porophyllum. These organs were dehydrated in an oven (CRAFT ® , Ciudad de México, Mexico) at 50 • C for three days; they were then left at room temperature (25 • C ± 2.0) for five days [19] and ground using a conventional blender. The extracts were obtained by maceration [20]; 15 g of the dry and ground material were mixed with 100 mL of absolute ethanol (EtOH, J.T. Baker ® ) or 100 mL of ethyl acetate (AcOEt, J.T. Baker ® ) to obtain each extract. Each mixture was placed in separate 200 mL beakers, which were kept in the dark and left to stand for 120 h at room temperature (25 • C ± 2.0). After this time, the extracts were filtered (Whatman ® filter paper no. 5) and dried at 50 • C in a rotary evaporator (Brinkmann/Büchi ® ). They were then weighed and all the extracts were re-dissolved in EtOH, adjusted to a concentration of 100 mg/mL and stored at 4 • C until further use.
The crude extracts with the highest antifungal activity were fractionated by thin layer chromatography (TLC) and column chromatography (CC). Thin layer chromatography was carried out using a mixture of hexane/ethyl acetate/methanol 80:20:1 v/v (J.T. Baker ® ), on aluminum plates precoated with silica gel 60 F254 (3.5 cm × 9 cm; Whatman ® ). After chromatography, the plates were left to dry outdoors and exposed to UV light (SPECTROLINE ® , Spectronics Corporation, Westbury, New York, NY, USA). Finally, the plates were revealed with 10% sulfuric acid (Fermont ® , Monterrey, NL, Mexico) to observe the bands [21].
Column chromatography was carried out using a glass column (50 cm in length) packed with 120 g of silica gel (Sigma ® , St. Louis, MO, USA). The extract was eluted with a mixture of hexane-ethyl acetate-methanol 80:20:1 (v/v). The collected fractions were analyzed by TLC to verify their successful separation and each fraction was concentrated with nitrogen (N 2 ) to 1 mL and stored at 5 • C.

Antifungal Activity Assays
A total of 12 extracts were obtained. Each extract was assayed with 0.5, 1, 2 and 5 mg/mL. The effect of the crude extracts on the mycelial growth of the fungi isolated from blueberry was evaluated according to the method proposed by Sánchez-Pérez et al. [22]. Each concentration was added to Petri dishes with PDA culture medium; 30 min after to allow the evaporation of EtOH and the diffusion of the extract, a disc of 5 mm in diameter with PDA and mycelium of the corresponding strain was placed in the center of the dish, which was then incubated at 28 • C. In addition, we included an absolute control (PDA + fungus), a negative control (PDA + fungus + EtOH) and a positive control (PDA + fungus + Thiabendazole 5 mg/mL, TECTO 60 ® wettable powder, 600 g IA kg −1 ). All bioassays were performed in triplicate. The effect of extracts and fractions was determined by measuring the diameter of the colonies with a digital vernier (Fisher Scientific, Waltham, MA, USA), and expressed as the percentage inhibition of mycelial growth compared to the negative control, according to the formula: % inhibition = average mycelial growth of the control-average mycelial growth of the treatment/average mycelial growth of the control × 100 [23].

Identification of Bioactive Secondary Metabolites
The fractions with the strongest antifungal effect were analyzed by gas chromatographymass spectrometry (GC-MS) using the fragrance method [24]. From each sample, 1 µL was analyzed with an Agilent 6890 (Agilent Technologies, Santa Clara, CA, USA) Gas Chromatography equipment with an HP-5MS (5% Phenyl 95% dimethylpolysiloxane) capillary column (30 m × 0.25 mm with 0.25 µm film thickness), which was coupled to an Agilent 5973N selective mass detector. Helium was used as a carrier gas at 7.67 psi with a 1.0 mL min −1 constant flow. The front inlet was maintained at 280 • C in a split ratio of 50:1. The initial oven temperature was set at 50 • C, which was increased to 280 • C at a rate of 5 • C min −1 and then held for 1 min at this constant temperature, subsequently increased to 380 • C at a rate of 25 • C min −1 and finally held at the temperature of 380 • C for 3 min. The mass spectrometer was operated in electrical ionization mode (EI), with a flow of 1 mL min −1 , 70 eV ionization voltage, the interface temperature at 300 • C and a scan range of 50-500 m/z. The compounds were identified by comparing the mass spectra of each constituent with those stored in the NIST02.L database. We only accepted peaks with a degree of purity of one, and the spectra were identified using a concordance threshold of 90%. The relative abundance of each compound was estimated by dividing its peak area by the sum of the areas of all peaks detected in the sample (total peak area).

Statistical Analysis
All treatments were arranged in a completely randomized design. The data of percent inhibition of mycelial growth were transformed with the arcsine: √ percent inhibition/100 [25]. Analysis of variance (p ≤ 0.05) and Tukey's test (p ≤ 0.05) were performed using the SAS statistical package (V. 9.0., SAS Inst. Inc., Cary, NC, USA).

Identification of Pathogenic Fungi of Blueberry and Pathogenicity Tests
Three pathogens of blueberry were identified at the molecular level: Pestalotiopsis clavispora, Colletotrichum gloeosporioides and Lasiodiplodia pseudotheobromae. The three fungi inoculated into blueberry plants caused dark-colored lesions on the stems, loosening of the epidermis and brown coloration of the leaf-tips (cancers and dieback disease).
The fungi were reisolated and identified from the lesions they caused to the plants, fulfilling Koch's postulates.

Antifungal Effect In Vitro of Crude Extracts on the Mycelial Growth of Blueberry Pathogens
The mycelial growth of blueberry pathogens was inhibited by at least one of the extracts from the plants under study. In the case of the treatments with L. hirta, the extract M5L (at a concentration of 5 mg/mL) inhibited 100% of the growth of P. clavispora and C. gloeosporioides, the same results obtained with the positive controls (Figure 1(A1,A2,E1,E2), Table 1).
The inhibitory activity of this extract against L. pseudotheobromae was only 22%, but it was significant (p ≤ 0.05) compared to the negative control (Figure 1(A3); Table 1). Other less effective extracts were M8Fl, which inhibited 24% of the growth of P. clavispora, and M1L, which inhibited 55% of the growth of C. gloeosporioides. A lower concentration of M5L (2 mg/mL) inhibited 21% of the growth of C. gloeosporioides (Table 1).
Regarding the treatments with A. porophyllum, the extract with the highest inhibitory activity was M4LS at a concentration of 5 mg/mL, inhibiting 100% of the growth of the three phytopathogens, the same result obtained with positive controls (Figure 1(C1-C3,E1-E3); Table 1). M4LS also showed significant inhibition (p ≤ 0.05) of L. pseudotheobromae at concentrations of 1 and 2 mg/mL (78.7 and 82.7%, respectively). At a concentration of 5 mg/mL, the M2LS extract inhibited 100% of C. gloeosporioides, whereas at 2 mg/mL it inhibited only 18%, with significant differences compared to the negative control. The M7L extract (5 mg/mL) was not very effective against P. clavispora and C. gloeosporioides. None of the extracts (L. hirta, A. ochroleuca and A. porophyllum) was effective against the mycelium of blueberry fungi at doses of 0.5 mg/mL (Table 1).

Identification of Pathogenic Fungi of Blueberry and Pathogenicity Tests
Three pathogens of blueberry were identified at the molecular level: Pestalotiopsis clavispora, Colletotrichum gloeosporioides and Lasiodiplodia pseudotheobromae. The three fungi inoculated into blueberry plants caused dark-colored lesions on the stems, loosening of the epidermis and brown coloration of the leaf-tips (cancers and dieback disease). The fungi were reisolated and identified from the lesions they caused to the plants, fulfilling Koch's postulates.

Antifungal Effect In Vitro of Crude Extracts on the Mycelial Growth of Blueberry Pathogens
The mycelial growth of blueberry pathogens was inhibited by at least one of the extracts from the plants under study. In the case of the treatments with L. hirta, the extract M5L (at a concentration of 5 mg/mL) inhibited 100% of the growth of P. clavispora and C. gloeosporioides, the same results obtained with the positive controls ( Figure  1(A1,A2,E1,E2), Table 1).

Fractions of Crude Extracts and Identification of Bioactive Compounds
The crude extracts that showed 100% antifungal activity in vitro were fractionated and evaluated. Nine of 10 fractions of the M5L extract of L. hirta showed significant antifungal activity (p < 0.05) compared to the negative control; the F10 fraction showed the highest percentages of inhibition (44.3%) against C. gloeosporioides. Few fractions had effect against P. clavispora, as was the case of F6, with 11% inhibition ( Table 2).

Discussion
The diseases affecting blueberry crops in northwestern Michoacán, Mexico, cause significant losses in production. This study identified three fungi that are often isolated from plants with symptoms of dieback and cancers: P. clavispora, C. gloeosporioides and L. pseudotheobromae. The first two have already been reported in blueberry crops by other studies [26,27], but we did not find reports of L. pseudotheobromae in blueberry, although it has been reported in other crops [28,29].
Given the significant problems caused by these fungi in the cultivation of blueberry, the use of plant extracts from medicinal plants has been considered as a control alternative [30]. This study shows that extracts of L. hirta, A. ochroleuca and A. Porophyllum have antifungal activity in vitro. Among the best treatments, the inhibitory effect (100%) of the extract M5L (AcOEt) of L. hirta on P. Clavispora and C. gloeosporioides was most prominent. Another study showed that extracts of other species of the same genus (L. camera) have an antifungal effect against F. oxysporum but not against C. gloeosporioides [8]. We found no reports on the antifungal effect of extracts of L. hirta against these or other pathogens of blueberry.
Another extract based on A. ochroleuca that inhibited 100% of the growth of both pathogens (at a concentration of 5 mg/mL) was M6LFr (AcOEt). These results are important because the antifungal effect of this extract was similar to that of the commercial fungicide Thiabendazole, which was used as positive control. Other studies have noted the antifungal effect of methanol extracts of A. ochroleuca against Fusarium sporotrichum ATCC 3299, Aspergillus niger, Trichophyton mentagrophytes and F. moniliforme [14].
The M4LS extract (AcOEt) of A. porophyllum was the only one that inhibited 100% of the three pathogenic fungi of blueberry, including L. pseudotheobromae, which was the most resistant fungus in this study. This is the first report of the antifungal effect of extracts of A. porophyllum against phytopathogenic fungi.
To carry out a preliminary approach to the chemical nature of the compounds present in fractions extracts with the highest antifungal activity, a GC-MS analysis was performed. The major compounds in the active fractions of the M5L extract of L. hirta were the diterpene Phytol and the triterpene α-Sitosterol. Phytol is a common compound in extracts of plants such as Porophyllum linaria [31], Vitex negundo [32], Rhaponticum acaule [33] and Apium nodiflorum [34]. It has also been reported as one of the main components of the essential oil of A. nodiflorum, which showed antifungal activity against yeasts, dermatophytes, and Aspergillus spp. [34]. The triterpene α-sitosterol has been isolated from Leucas aspera [35].
The compound identified in the most active fraction of the M6LFr extract of A. ochroleuca was Benzene, 1,3-bis (3-phenoxyphenoxy)-. The major compound in essential oil of Bridelia micrantha, Benzene, 1,3-bis (3-phenoxyphenoxy)-has shown antimicrobial activity against Mycobacterium tuberculosis [36]. However, there are no reports of this compound having antifungal activity against P. clavispora and C. gloeosporioides.
A single compound was identified in the three most active fractions from the M4LS extract of A. porophyllum: Hexanedioic acid, bis (2-ethylhexyl) ester. This compound has been isolated from the roots of Stellera chamaejasme [37], the stem of Hugonia mystax [38], and even from Streptomyces sp. [39]. This compound has shown antifungal activity against Monilinia fructicola [37] and Fusarium sp. [39]. However, it is necessary to carry out experiments that lead us to compounds purification and confirm their antifungal activity.

Conclusions
This study shows that extracts of the medicinal plants L. hirta, A. ochroleuca and A. porophyllum inhibited 100% of the mycelial growth in vitro of P. clavispora, C. gloeosporioides and L. pseudotheobromae at a concentration of 5 mg/mL. Analysis by GC-MS indicates that the bioactive compounds present in the most effective extracts are the diterpene Phytol; the triterpenes α-Sitosterol; and Toluene, Benzene, 1,3-bis(3-phenoxyphenoxy)-and Hexanedioic acid, bis(2-ethylhexyl) ester. This demonstrates the potential of L. hirta, A. ochroleuca and A. porophyllum as sources of antifungal compounds.

Data Availability Statement:
The authors declare that the data supporting the findings of this study are available within the article.