Strawberry Anthracnose Research Articles

Bacillus amyloliquefaciens strains control strawberry anthracnose through antagonistic activity and plant immune response intensification

Anthracnose disease caused by Colletotrichum gloeoesporioides can have devastating effects on strawberry production. In addition to the use of fungicides, feasible strategies for controlling this disease include the application of beneficial microorganisms. In this study, we assayed the effects of two Bacillus amyloliquefaciens strains, PMB04 and PMB05, on the control of strawberry anthracnose. First, an in vitro inhibitory assay revealed that the hyphal growth of C. gloeosporioide SC01 was inhibited by PMB04 more strongly than by PMB05. We further found that the culture filtrate from PMB04 was lethal to the conidia of SC01. Meanwhile, PMB05 was previously demonstrated to be a rhizobacterium that intensifies plant immunity in response to bacterial elicitors. Thus, its effect on plant immune signals, such as the rapid reactive oxygen species (ROS) generation and callose deposition triggered by the conidia of SC01 on strawberry leaves, were evaluated in this study. The results showed that only the PMB05 strain could intensify the plant immune signals activated by SC01. In an inoculation assay, meanwhile, both strains exhibited superior efficacy in reducing the occurrence of anthracnose on strawberry seedlings and fruits. More specifically, PMB04 and PMB05 showed better ability to control anthracnose on fruits and seedlings, respectively. We concluded that both the lethal effect on spores caused by PMB04 and the intensification of plant immunity caused by PMB05 could be key factors in controlling strawberry anthracnose. More interestingly, we present a case in which a plant immune response-intensifying strain was applied to control strawberry anthracnose.

Antifungal Activity of Serratia rubidaea Mar61-01 Purified Prodigiosin Against Colletotrichum nymphaeae, the Causal Agent of Strawberry Anthracnose

Strawberry anthracnose, caused by Colletotrichum nymphaeae is an important disease of strawberry. In this study, an endophytic bacterium isolated from the stem of Fragaria × ananassa cv Paros and identified as Serratia rubidaea strain Mar61-01 based on phenotypic, biochemical characteristics and molecular phylogenetic analysis. Antagonistic activities of this endophytic bacterium against C. nymphaeae were investigated under in vitro, in vivo and greenhouse conditions. The strain Mar61-01 reduced mycelial growth and conidial germination of C. nymphaeae in in vitro tests. Furthermore, it reduced disease severity on inoculated strawberry fruits and seedlings compared with uninoculated control. In addition, the strain Mar61-01 produced prodigiosin pigment. The pigment was purified by thin layer chromatography and its chemical structure was characterized by FT-IR and NMR (400 MHz) spectra. The results indicated that prodigiosin is a key feature in biocontrol of C. nymphaeae. The inhibition growth of C. nymphaeae under in vitro, in vivo, and greenhouse conditions showed that S. rubidaea Mar61-01 has the potential for managing of strawberry anthracnose.

First Report of Xanthomonas fragariae Causing Angular Leaf Spot on Strawberry (Fragaria x ananassa) in Taiwan.

Angular leaf spot of strawberry, considered an A2 quarantine pest by the European and Mediterranean Plant Protection Organization (EPPO 2019), is an important bacterial disease in many regions. Since 2017, symptoms similar to angular leaf spot were observed in several strawberry cultivars including 'Taoyuan No. 1' and 'Xiang-Shui'. Early symptoms were angular, water-soaked lesions on the abaxial leaf surface, and later, reddish-brown irregular spots and coalesced lesions developed on the adaxial surface. In the humid conditions, sticky bacterial ooze exuding from lesions was observed. To isolate the causal agent, leaves showing water-soaked lesions were surface sterilized, cut into small pieces and soaked in 5 ml sterile water for at least 15 min. The supernatant from the cut-up pieces was serially diluted followed by spreading on sucrose peptone agar (SPA) (Hayward 1960). After incubating at 20°C for 4-5 days, single colonies grown on SPA were transferred to a new SPA plate and cultured at 20°C until colonies appeared. The yellow, glossy and mucoid colonies, which resembled the colony morphology of Xanthomonas fragariae, were selected as candidates for further confirmation. First, bacterial DNA of four candidate isolates, B001, B003 and B005 from Miaoli County and B004 from Taoyuan City, was PCR amplified with X. fragariae-specific primers: XF9/XF12 (Roberts et al. 1996) and 245A/B and 295A/B (Pooler et al. 1996). All four isolates could be detected by XF9/XF12 primer. Furthermore, isolates B003 and B004 could be detected by both 245A/B and 295A/B primers, while B001 and B005 could be detected by 295A/B only. Next, DNA gyrase subunit B (gyrB) was PCR amplified with the primers XgyrB1F/XgyrB1R (Young et al. 2008). The gyrB sequences of these four isolates were deposited in GenBank with accession numbers MT754942 to MT754945. The gyrB phylogenetic tree was constructed based on Bayesian inference analysis and maximum likelihood analysis. The gyrB sequences of the four isolates from Taiwan clustered in the clade containing the type strain of X. fragariae ICMP5715, indicating that they belong to X. fragariae. B001 and B005 formed a sub-group separated from B003 and B004, suggesting genetic differences between these isolates. To fulfill Koch's postulates, the abaxial surface of strawberry leaves were syringe infiltrated (KJP Silva et al., 2017) or wounded inoculated (Wang et al., 2017) with bacterial suspensions (final OD600 = 1.0-2.0) prepared from colonies of B001 and B003 washed from SPA plates. Inoculated plants were enclosed in a plastic bag (> 90% RH) at 25/20°C (day/night) under a 12-h/12-h photoperiod. After 7-14 days, water-soaked lesions similar to those observed in the field were developed on all inoculated leaves. The bacteria were successfully re-isolated from lesions of inoculated leaves and confirmed by specific primers XF9/XF12, 245A/B and 295A/B. We also found that the disease commonly occurs in the strawberry fields/nurseries with sprinkler irrigation during winter or early spring, and was particularly serious in the windward side or near riverside. To our knowledge, this is the first report of X. fragariae causing angular leaf spot on strawberry in Taiwan. Currently, the disease only occurs severely in certain regions, but establishment of effective management strategies will be needed to prevent spreading of this disease and potential economic loss in the future.

Biocontrol efficacy and possible mechanism of Streptomyces sp. H4 against postharvest anthracnose caused by Colletotrichum fragariae on strawberry fruit

Anthracnose is a fungal disease caused by Colletotrichum species, which is detrimental to numerous fruits, including strawberry. Use of fungicides to maintain fruit quality leads to potential environmental pollution and health risk. Biocontrol using beneficial microorganisms such as Streptomyces has been applied successfully for controlling postharvest diseases of fruit. In this study, strain H4 with a high antifungal activity against C. fragariae was isolated from Dichotella gemmacea in Xisha islands of South China Sea. Combining the morphological and biochemical characteristics with the 16S rRNA sequence analysis, this strain was assigned as Streptomyces sp. A preventive treatment using strain H4 extracts significantly reduced severity and incidence of anthracnose disease and maintained fruit hardness and color on harvested strawberry fruits. A minimum inhibitory concentration (MIC) and a minimum fungicidal concentration (MFC) were 1.563 × 10−3 g L-1 and 3.125 × 10−3 g L-1, respectively. Extracts could effectively inhibit mycelial growth and spore germination of C. fragariae in vitro. The mycelial structure of pathogenic fungi showed deformation, shrinkage, collapse and tortuosity. A significant decrease in sugar and protein contents was also observed in treated C. fragariae mycelia. Fourteen chemical compounds were identified by gas chromatography-mass spectrometer (GC–MS). Dibutyl phthalate was the major constituent. Notably, strain H4 and its extracts exhibited a broad-spectrum antifungal activity against seven selected plant pathogenic fungi. Hence, Streptomyces sp. H4 and its metabolites have a high efficiency of antagonistic roles against phytopathogenic fungi diseases. It provides a promising biological agent to control anthracnose of strawberry fruit.

Furanocoumarin with Phytotoxic Activity from the Leaves of Amyris elemifera (Rutaceae).

Bioassay-guided fractionation of the ethyl acetate extract of Amyris elemifera leaves was carried out to identify phytotoxic and antifungal constituents. A novel phytotoxic furanocoumarin 8-(3-methylbut-2-enyloxy)-marmesin acetate (1) and its deacyl analog 8-(3-methylbut-2-enyloxy)-marmesin (2) were isolated. The X-ray crystal structure determination is reported for the first time for 1. Both 1 and 2 have the S configuration at C-2′ based on X-ray crystallographic data. Both these compounds inhibited the growth of the dicot Lactuca sativa (lettuce) and the monocot Agrostis stolonifera with a more pronounced inhibitory effect on the monocots at 330 μM by 1. In Lemna paucicostata Hegelm phytotoxicity bioassay, the IC50 value for 1 was 26 μM, whereas 2 had an IC50 value of 102 μM. Compounds 1 and 2 were weakly antifungal against Colletotrichum fragariae Brooks in TLC bioautography.

Characterization of Colletotrichum spp. Sensitivity to Carbendazim for Isolates Causing Strawberry Anthracnose in China.

Strawberry anthracnose caused by Colletotrichum species is an important disease that may cause significant economic losses. Based on multilocus sequence analyses and morphological characteristics, 64 isolates from strawberry anthracnose samples collected from nine Chinese provinces and municipalities were identified as three species: Colletotrichum fructicola (29 isolates), Colletotrichum siamense (23 isolates), and Colletotrichum nymphaeae (12 isolates). Isolates of C. siamense showed strong aggressiveness to fruit and leaves. Isolates of C. fructicola showed strong aggressiveness to crowns. Isolates of C. nymphaeae were weakly or not pathogenic to fruit, leaves, or crowns. Sensitivity to carbendazim was determined for a total of 75 isolates, including 11 previously preserved. Two isolates of C. siamense were highly resistant (HR) and 21 were moderately resistant (MR). Nine isolates of C. fructicola were sensitive (S), 24 were HR, and four were MR. All 15 isolates of C. nymphaeae were insensitive, and their mycelial growth was not completely inhibited on potato dextrose agar amended with 500 μg/ml carbendazim. Beta-tubulin (TUB2) of representative isolates was amplified and sequenced, revealing a glutamic acid substituted by alanine at codon 198 in HR isolates of C. siamense and C. fructicola. MR isolates of C. siamense and C. fructicola had a point mutation at codon 200, causing a replacement of phenylalanine acid by tyrosine. No point mutation was detected at codons 50, 167, 198, 200, or 240 in TUB2 of C. nymphaeae insensitive isolates. Overall, this study revealed that C. fructicola was the dominant species causing anthracnose on strawberry and could improve the understanding of the management of fungicide resistance in Colletotrichum species on strawberry in China.

Volatile compounds mediated effects of Stenotrophomonas maltophilia strain UN1512 in plant growth promotion and its potential for the biocontrol of Colletotrichum nymphaeae

A novel endophytic bacterium, designated strain UN1512, was isolated from strawberry leaves, and its antifungal activity against Colletotrichum nymphaeae was estimated under in vitro, in vivo, and greenhouse conditions. The bacterial isolate was identified as Stenotrophomonas maltophilia via morphological, biochemical, and molecular traits. The strain UN1512 decreased the mycelial growth of C. nymphaeae (54.92%) in dual culture. The volatile and non-volatile compounds produced by strain UN1512 reduced mycelial growth of fungal pathogen by 17.85% and 30.06%, respectively. Also, the strain was able to secrete of protease, chitinase, pectinase, siderophore, IAA, and gibberellin. In addition, disease severity of strawberry anthracnose was inhibited via inoculating the bacterial suspension in phylloplane, rhizosphere, and healthy fruits. Also, the effect of volatile compounds (VOCs) produced by strain UN1512 on growth promotion of tomato plants was investigated under in vitro and growth chamber experiments. The results showed that, these compounds significantly enhance the fresh weight and length of roots and shoots. The VOCs produced by strain UN1512 were analyzed by GC-MS and 31 identified VOCs, that Benzothiazole (18.271%), 1,3,5,7-Cyclooctatetraene-1-carboxaldehyde (9.308%), Carbonic acid, octadecyl phenyl ester (5.203%), Benzaldehyde, 2,5-bis[(trimethylsilyl)oxy]- (4.086%), Estragole (3.557%), Benzaldehyde (3.506%) were the major components. Our studies report that strain UN1512 was capable to inhibit the causal agent strawberry anthracnose, and the VOCs produced by this strain increased the tomato seedlings growth.

Diversity and pathogenicity of Colletotrichum species causing strawberry anthracnose in Taiwan and description of a new species, Colletotrichum miaoliense sp. nov.

Strawberry is a small fruit crop with high economic value. Anthracnose caused by Colletotrichum spp. poses a serious threat to strawberry production, particularly in warm and humid climates, but knowledge of pathogen populations in tropical and subtropical regions is limited. To investigate the diversity of infectious agents causing strawberry anthracnose in Taiwan, a disease survey was conducted from 2010 to 2018, and Colletotrichum spp. were identified through morphological characterization and multilocus phylogenetic analysis with internal transcribed spacer, glyceraldehyde 3-phosphate dehydrogenase, chitin synthase, actin, beta-tubulin, calmodulin, and the intergenic region between Apn2 and MAT1-2-1 (ApMAT). Among 52 isolates collected from 24 farms/nurseries in Taiwan, a new species, Colletotrichum miaoliense sp. nov. (6% of all isolates), a species not previously known to be associated with strawberry, Colletotrichum karstii (6%), and three known species, Colletotrichum siamense (75%), Colletotrichum fructicola (11%), and Colletotrichum boninense (2%), were identified. The predominant species C. siamense and C. fructicola exhibited higher mycelial growth rates on potato dextrose agar and caused larger lesions on wounded and non-wounded detached strawberry leaves. Colletotrichum boninense, C. karstii, and C. miaoliense only caused lesions on wounded leaves. Understanding the composition and biology of the pathogen population will help in disease management and resistance breeding.

Preharvest and Postharvest Application of Garlic and Rosemary Essential Oils for Controlling Anthracnose and Quality Assessment of Strawberry Fruit During Cold Storage.

This study assessed the feasibility of using essential oils (EOs) against Colletotrichum nymphaeae inciting strawberry anthracnose. Two EOs, extracted from Allium sativum (garlic) and Rosmarinus officinalis (rosemary), were selected because their fungicide efficacy was already well characterized under laboratory and greenhouse conditions. We characterized both EOs in terms of efficacy and impact on qualitative traits and sensory quality of strawberry fruit. The gas chromatography–mass spectrometry analysis confirmed the Diallyl trisulfide (29.08%) and (α)-pinene (15.779%) as the main components of A. sativum and R. officinalis EOs, respectively. Both A. sativum and R. officinalis EOs significantly inhibited the mycelial growth and conidial germination of C. nymphaeae in contact and vapor assays compared with untreated control. However, EC50 assay indicated A. sativum EO was more effective than R. officinalis EO against the pathogen. Malformations of the vegetative structures of the pathogen exposed to both EOs were revealed as shriveled, collapsed, and swelling mycelia in the cultures. Both EOs confirmed their efficacy under in vivo and greenhouse conditions; in fact, they significantly reduced the development of fruit decay and anthracnose disease incidence and severity, compared with untreated controls. Both EOs preserved sensory attributes and quality parameters of strawberry fruit including firmness, total soluble solids, ascorbic acid, antioxidant activity, and anthocyanin, but may leave unwanted smells. These findings suggest that two EOs can potentially represent an alternative to synthetic chemical fungicides against C. nymphaeae preserving fruit quality factors, although their cost and the impact on the fruit odor must be carefully taken into consideration before developing a commercial product.

Identification of antagonistic bacteria Bacillus velezensis JK3 against anthracnose of strawberry and its antipathogenic activity

Identification of antagonistic bacteria Bacillus velezensis JK3 against anthracnose of strawberry and its antipathogenic activity

Characterization and Fungicide Sensitivity of Colletotrichum Species Causing Strawberry Anthracnose in Eastern China.

Strawberry anthracnose caused by Colletotrichum spp. is one of the most serious diseases in the strawberry fields of China. In total, 196 isolates of Colletotrichum were obtained from leaves, stolons, and crowns of strawberry plants with anthracnose symptoms in eastern China and were characterized based on morphology, internal transcribed spacer (ITS), and β-tubulin (TUB2) gene sequences. All 196 isolates were identified as the Colletotrichum gloeosporioides species complex. In total, 62 strains were further identified at the species level by phylogenetic analyses of multilocus sequences of ITS, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat), calmodulin (CAL), and TUB2. Three species from the C. gloeosporioides species complex were identified: Colletotrichum siamense, C. fructicola, and C. aenigma. Isolates of C. siamense were tolerant to high temperatures, with a significantly larger colony diameter than the other two species when grown above 36°C. The inoculation of strawberry plants confirmed the pathogenicity of all three species. C. siamense isolates resulted in the highest disease severity. The in vitro sensitivities of C. siamense and C. fructicola isolates to azoxystrobin and three demethylation-inhibitor (DMI) fungicides (difenoconazole, tebuconazole, and prochloraz) were determined. Both species were sensitive to DMI fungicides but not to azoxystrobin. C. siamense isolates were more sensitive to prochloraz, while C. fructicola isolates were more sensitive to difenoconazole and tebuconazole. The present study provides valuable information for the effective management of strawberry anthracnose.

FaRCa1 Confers Moderate Resistance to the Root Necrosis Form of Strawberry Anthracnose Caused by Colletotrichum acutatum

FaRCa1 is a major locus conferring resistance to anthracnose fruit rot (AFR) caused by Colletotrichum acutatum, an important pathogen of strawberry (Fragaria ×ananassa). The objective of this study was to characterize the effects of FaRCa1 on anthracnose root necrosis (ARN) via root inoculations and DNA marker characterization of the locus. A subgenome-specific high-resolution melting (HRM) marker for an insertion/deletion (InDel) near FaRCa1 was designed using the ‘Camarosa’ octoploid reference genome. The marker was used to genotype cultivars and advanced selections studied in two seasons. A root disease screening method was developed in which roots were cut and dipped in a spore suspension before planting, using a mixture of three local isolates of the C. acutatum species complex. ARN was indirectly scored by calculating image-based leaf area differences among inoculated and noninoculated plants. The allele of FaRCa1 conferring resistance to AFR also conferred a significant reduction in ARN. Thus, a robust and easily scored DNA test is now available to breeders for selecting for resistance to both the fruit and root forms of strawberry anthracnose.

Genetic Diversity of Colletotrichum spp. Causing Strawberry Anthracnose in Zhejiang, China.

Anthracnose is a serious fungal disease that primarily infects strawberry roots and stolons during development. Here, 91 isolates from different areas of Zhejiang province, China, were collected. Morphological characteristics were analyzed, and a phylogenetic analysis based on multiple genes (actin, internal transcribed spacer, calmodulin, glyceraldehyde-3-phosphate dehydrogenase, and chitin synthase) was performed. We found that all of the Colletotrichum species causing strawberry anthracnose belonged to the Colletotrichum gloeosporioides complex. Among them, we identified 48 isolates of C. fructicola, 21 isolates of C. siamense, 13 isolates of C. gloeosporioides, and 9 isolates of C. aenigma. C. siamense was distributed in the central and eastern regions of Zhejiang province (Hangzhou, Jinhua, Shaoxing, Ningbo, and Taizhou). This is the first report of C. siamense causing strawberry anthracnose in Zhejiang province. C. fructicola was the most dominant species causing strawberry anthracnose in Zhejiang province. We identified the four species causing strawberry anthracnose in Zhejiang province, which will improve our understanding of the strawberry anthracnose epidemic and will benefit the development of future control measures.

Draft Genome Sequence of the Strawberry Anthracnose Pathogen Colletotrichum fructicola.

Colletotrichum fructicola is a causal agent of strawberry anthracnose and a major economic pathogen of horticultural and ornamental crops worldwide. Here, we present an annotated draft genome sequence for a C. fructicola isolate previously used for transcriptomic analysis. The assembly totals 58.0 Mb in 477 contigs with 18,143 predicted genes.

A novel strain of endophytic Streptomyces for the biocontrol of strawberry anthracnose caused by Glomerella cingulata

Anthracnose caused by Glomerella cingulata is one of the most devastating diseases of strawberry in Japan, particularly during its nursery period in the summer. In this study, we aimed to isolate and screen endophytic actinobacteria, to identify potential biocontrol agents capable of suppressing strawberry anthracnose. A total of 226 actinobacteria were successfully isolated from surface-sterilized strawberry tissues. In the first screening, 217 out of 226 actinobacteria isolates were studied for their suppression effect on strawberry anthracnose using a detached leaflet assay. It was discovered that isolates MBFA-172 and MBFA-227 markedly suppressed the development of anthracnose lesions. The efficacy of both isolates was then tested on two-month-old strawberry plug seedlings in a controlled environmental chamber. It was found that isolate MBFA-172 provided consistent disease suppression and was thus selected as a final candidate for further evaluation in a glasshouse experiment. Results showed that the severity as well as incidence rate of strawberry anthracnose was significantly reduced by treatment with isolate MBFA-172 compared with that of untreated control. Accordingly, the disease control efficacy provided by MBFA-172 was statistically comparable to the chemical fungicide propineb. A re-isolation experiment using a spontaneous thiostrepton-resistant mutated strain of isolate MBFA-172 revealed that it efficiently colonized the above-ground tissues of strawberry plants for at least three weeks after spray treatment. Using cultural, morphological, and physiological tests combined with 16S rRNA-based molecular analysis, MBFA-172 was identified as a moderately thermophilic Streptomyces thermocarboxydus-related species. Upon review, our results strongly indicated that MBFA-172 is a promising biocontrol agent for strawberry anthracnose.

First Report of Botrytis cinerea Causing Leaf Spot on Strawberry in California

On 29 October 2018, a leaf spot disease was observed on strawberry leaves (Fragaria × ananassa Duch., cultivar Portola) on organically grown plants in high plastic tunnels in Santa Maria, California. Symptoms consisted of circular lesions measuring 2 to 3 cm in diameter, concentric rings with a darker brown outer ring and fungal sporulation in the center of the lesion. The diseased leaf sample was surface sterilized in 10% sodium hypochlorite for 1 min, rinsed thrice in sterile water, and placed on potato dextrose agar (PDA) after drying. The resulting culture produced conidia that were hyaline and round to ellipsoid (8 to 14 × 5 to 8 µm, n = 50) and sclerotia that were hard, dark, round to oblong, and measured 2 to 12 × 2 to 9 mm (n = 50). Based on its morphological characteristics in culture, the isolate was putatively identified as Botrytis cinerea (Hong et al. 2001; Jarvis 1977). The isolate produced an average of 133 sclerotia per Petri dish (100 × 15 mm; n = 3) with PDA, an uncommon feature of B. cinerea isolates from strawberry. Koch’s postulates were performed using two inoculation methods: (i) Botrytis agar plug on leaf; and (ii) conidial suspension on leaf. Leaf inoculations used a spore suspension containing 1 × 10⁶ conidia/ml and were replicated three times. Leaf inoculations using an agar plug or spore suspension were allowed to dry, misted with water, and covered with a plastic bag to maintain continuous moisture. Agar-plug inoculated leaves produced symptoms in 4 days, whereas the spore-suspension inoculations produced symptoms in 9 days. Leaf symptoms matched those observed in the field. The pathogen was reisolated from symptomatic tissue, and its morphological features matched those of the original culture. In both inoculation methods, the noninoculated controls did not result in any disease symptoms. Cross pathogenicity to leaves using an isolate of B. cinerea collected from fruit was successful in causing foliar disease, but symptoms lacked concentric rings. To determine the fungal species, total genomic DNA of five subcultures of the original field isolate was extracted using a DNeasy Plant Mini Kit (Qiagen). The heat shock protein (HSP60) (Hsp60for+/Hsp60 rev+ primers), RNA polymerase II-binding (RPB2) (RPB2for+/RPB2 rev+ primers), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (G3PDHfor+/G3PDH rev+ primers) genes were amplified (Staats et al. 2004). The PCR amplicons were purified and sequenced. The sequences were processed using BLAST in the National Center for Biotechnology, and results showed 100% homology with the HSP60 (MH796663), RPB2 (MH479932), and G3PDH (MH479930) sequence for isolates of B. cinerea. The sequences were deposited in GenBank with accession numbers MK919494 (HSP60), MK919495 (RPB2), and MK919496 (G3PDH). B. cinerea is a common pathogen of strawberry fruit and can cause gray mold, postharvest rot, and dry crown rot but has not been reported as causing a leaf spot. This disease is rare and considered of minor significance to strawberry production. However, it could be of greater significance in protected culture where humidity is higher. To our knowledge, this is the first report of B. cinerea causing strawberry leaf spot in California or elsewhere.

Design, Synthesis and Biological Evaluation of N-((2-phenyloxazol-4-yl)methyl) Pyrimidine Carboxamide Derivatives as Potential Fungicidal Agents

Abstract Twelve N-((2-phenyloxazol-4-yl)methyl) pyrimidine carboxamide derivatives were designed, synthesized, and characterized by 1H NMR, 13C NMR, and HRMS. The fungicidal activities of these new compounds against Sclerotinia sclerotiorum, Botrytis cinereal, and Colletotrichum fragariae were evaluated. The results indicated that compounds 5b, 5f, and 5g displayed potential fungicidal activities against tested fungi, especially 5f exhibited IC50 value of 28.9 mg/L against S. sclerotiorum. Moreover, the compounds 5f and 5g showed IC50 values of 54.8 mg/L and 62.2 mg/L against C. fragariae respectively, which shows that they were more active than the commercial fungicide hymexazol. The superficial structure-activity relationships were discussed, which may be of benefit for the development of fungicides and discovery of novel fungicides.

APPLICATION OF SALVIA OFFICINALIS AND PICEA ABIES ESSENTIAL OILS FOR CONTROLLING COLLETOTRICHUM SPP

Colletotrichum spp. is a significant strawberry fruit pathogen, causing yield losses of up to 80% - growing resistance to pesticides demands to new, environmentally-friendly plant protection. Essential oils (EO) are one of the biological plant protection products suitable for pathogens control. The antimicrobial and antifungal activity of EO, biodegradability and low toxicity, make it potential for use in plant protection against pathogens instead of chemicals. The present study was carried out to investigate the antifungal effects of Salvia officinalis and Picea abies essential oils against strawberry Colletotrichum spp. The research carried at the LAMMC Institute of Horticulture. The bio-fungicidal effect was assessed based on radial growth inhibitions. There were evaluated several EO concentrations: 1000-1800 µl/l. Single-spore isolate fragment placed in the centre of PDA with different concentrations. Plates were incubated at oils 25 o C in darkness and evaluated after 2, 4, 7 days. S. officinalis and P. abies EO showed inhibitory effect on Colletotrichum spp. mycelial development. The S. officinalis inhibitory effect was more than 50% in all concentrations. The present study revealed that highest 1800 μl/l S. officinalis EO concentration Colletotrichum spp. colony diameter was significantly lower (1.84 cm) compared with control (5.75 cm). However, P. abies inhibition more than 50 % were only in concentrations from 1600 μl/l. According to our results, treatment with EO can reduce the growth of Colletotrichum spp. and EO could be an effective potential bio-fungicide to control strawberry anthracnose. EO as bio-fungicides characteristics is their natural origin and low risk for resistance development.

Characterization of Est-SSR markers related to Colletotrichum fructicola infection in strawberry (Fragaria ×ananassa Duchase)

Colletotrichum fructicola causes strawberry anthracnose, which results in considerable economic losses in quality and production in Asia. However, the molecular mechanism of strawberry infection with C. fructicola is still poorly understood. Here, we employed RNA-Seq technology to mine the strawberry transcriptome for EST-SSRs (Expressed Sequence Tag–Simple Sequence Repeats) following C. fructicola infection. Fortunately, we detected 3019 potential EST-SSRs from the transcriptomic data of cv. Jiuxiang. The seven commonly occurring repeat motifs were A/T (278), GAA/TTC (263), GAG/CTC (215), AAG/CTT(188), GGA/TCC (163), AGA/TCT (153) and AGG/CCT (153). We identified 31, 84 and 129 differentially expressed EST-SSR-containing sequences corresponding to 24, 72 and 96 Hpi, while 13 DE-EST-SSRs -containing sequences were differentially expressed and common for all the three time points. Furthermore, we identified allele polymorphisms for some DE-EST-SSRs among several resistant and susceptible strawberry varieties and wild species. The EST-SSRs in our research were related to the reported QTL resistance loci in strawberry which are differentially expressed after C. fructicola infection and have allele polymorphisms in different strawberry varieties and wild species. These provide a clue for utilisation of the EST-SSRs in marker-assisted breeding to improve anthracnose-resistant breeding in future.

Management of Strawberry Anthracnose Using Plant Essential Oils as Bio-fungicides, and Evaluation of Their Effects on Quality of Strawberry Fruit.

Colletotrichum nymphaeae is the causal agent of strawberry anthracnose, which is one of the most important disease affecting strawberry plant in Iran. This research aimed to apply the selected plant essential oils (EOs) such as Achillea millefolium, Mentha longifolia, and Ferula kuma to the management of strawberry anthracnose disease under in vitro, in vivo, and greenhouse conditions. In vitro tests indicated that all the EOs and fungicide were able to inhibit mycelial growth and conidial germination of the pathogen. Scanning electron microscopy (SEM) revealed that EOs significantly suppressed the mycelia growth and caused a change in morphology of fungal mycelia. The severity of strawberry anthracnose disease was significantly (p ≤ 0.05) reduced by all EOs under in vivo and greenhouse conditions. Results of all experiments showed that M. longifolia EO was the best EO to control C. nymphaeae. Also, EOs almost reduced weight loss and preserved firmness, ascorbic acid, total phenol, antioxidant activity (DPPH), and enzyme peroxidase activity in treated fruit. Moreover, EOs preserved the sensory quality of strawberry fruit during the storage period so that there were no significant differences between treatments (EOs) in their appearance, flavor, odor attributes, and overall evaluation compared to the control. Our results indicate that EOs are excellent bio-fungicides for the management of strawberry anthracnose.