Colletotrichum Acutatum Research Articles

First report of Colletotrichum fioriniae causing anthracnose on the fruit of omija (schisandra) in South Korea.

Omija or Schisandra (Schisandra chinensis Baillon) is one of Korea's most unique fruits and have been used in herbal medicine. It is also used for making cold drinks during summer and hot tea during winter. Among the different diseases, anthracnose poses great threat for omija production as it reduces the yield and market quality of fruits. In September 2021, severe anthracnose symptoms were observed in some commercial farmer fields (36°36'14.9"N 127°59'31.3"E) in Mungyeong and disease incidence was approximately 30 to 35%. The symptoms on fruits were manifested as small sunken lesions. The presumed fungi were isolated from necrotic tissues on potato dextrose agar (PDA) and purified using the single spore isolation technique (Cai et al. 2009; Hassan et al.2019). A total of 20 morphological similar isolates were obtained from 18 sampled fruits. The cultures were incubated at 25°C in the dark for 5 to 7 days. The colonies were pink with white aerial mycelium on the upper side and pink with black zones on the reverse. All the colonies produced fusiform conidia measuring 12.7 to 17.5 × 4.7 to 7.4 μm (n=50). The appressoria were brown, globose to ellipsoid and measuring 6.3 to 12 × 5.9 to 9.7 μm (n=30). Morphological characteristics of present isolates were in concordance with that of Colletotrichum acutatum sensu lato (Damm et al., 2012). For molecular identification, genomic DNA of five representative isolates was extracted for sequencing of target markers and the phylogenetic tree was constructed. For amplification and sequencing of internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT) chitin synthase-1 (CHS-1), histone 3 (HIS3) and beta-tubulin (TUB2), the primer pairs ITS1/ITS4, GDF1/GDR1, ACT-512F/ACT-783R, CHS1-79F/CHS-1-354R, CYLH3F /CYLH3R and BT2a /BT2b were used respectively (Damm et al., 2012). The resulting sequences were deposited in GenBank with accession numbers LC680890 to LC680914 and LC687627 to LC687631. The maximum likelihood tree based on concatenated sequences of the five genes shows that present isolates were grouped in the C. fioriniae. Pathogenicity on red schisandra fruits was confirmed using the two isolates OR1 and O59. Fruit clusters (20-30 fruits/ cluster) were surface sterilized by dipping them in 1% NaOCl for 3 minutes, rinsing in sterile distilled water, and placing them in a sterile plastic box containing moist paper tissues. All the fruits in each cluster were inoculated with 10 μl of 106 conidia/ml suspensions of each isolate by following either the wounded or un- wounded method (Hassan et al.2019). Fruit clusters treated with sterile distilled water served as a control. Three fruit clusters were inoculated per isolate for each method. With the wounding method, all the inoculated fruits showed typical anthracnose symptoms after 4 days of incubation at 25 ± 1°C in the dark, while the control and un- wounded fruits remained symptomless. This test was repeated twice. The species C. fioriniae were reisolated from the infected fruit. The morphology and ITS sequences of the reisolated fungi were consistent with the original one, fulfilling Koch's postulates. The species C. fioriniae have been described as a causal agent of anthracnose of numerous hosts worldwide including South Korea (Damm et al., 2012; Hassan et al.2019; Oo et al. 2016; Lee et al. 2020). To our knowledge, this is the first report of C. fioriniae as causal agent of omija anthracnose in South Korea. As this disease results in deterioration of fruits quality, management practices should be tailored to address C. fioriniae management. Acknowledgments This research was supported by Kyungpook National University Research Fund, 2021. References Cai, L., et al. 2009. Fungal Divers. 39:183. Damm, U., et al. 2012. Stud. Mycol. 73:37. Hassan, O., et al. 2019. Sci. Rep. 9:12089. Lee, D., et al. 2020. Mycobiology. 48: 210. Oo, M.M., et al. 2016. Mycobiology. 44: 325.

Susceptibility of apple fruits from selected cultivars to the Colletotrichum acutatum species complex

The pathogenicity of five isolates of Colletotrichum acutatum species complex (CASC) was tested on artificially injured apple fruits of the cultivar ‘Golden Delicious’ under laboratory conditions. Then, ten apple cultivars were tested for their relative susceptibility to CASC. Differences in the pathogenicity of the CASC isolates were confirmed in the test. The highest degree of pathogenicity was showed by the isolates from the strawberry and DCSBPM J1 from the apple. No cultivars were resistant to the pathogen. The disease incidence ranged from 57.5% to 100%. The individual cultivars showed different susceptibility to the tested isolates. The cultivars ‘Braeburn’, ‘Jonagold’ and ‘Rubinola’ showed the least susceptibility to the pathogen, while the cultivars ‘Jonagored’ and ‘Otava’ were recorded as the most susceptible to CASC. All the tested isolates were confirmed, by the conventional PCR method, to belong to CASC. The sequences of isolates DCSBPM J1 and DCSBPM 1209 were deposited in the GenBank.

Potentiality of actinobacteria to combat against biotic and abiotic stresses in tea [Camellia sinensis (L) O. Kuntze

Tea (Camellia sinensis (L) O. Kuntze) is a long-duration monoculture crop prone to several biotic (fungal diseases and insect pest) and abiotic (nutrient deficiency, drought and salinity) stress that eventually result in extensive annual crop loss. The specific climatic conditions and the perennial nature of the tea crop favour growth limiting abiotic factors, numerous plant pathogenic fungi (PPF) and insect pests. The review focuses on the susceptibility of tea crops to PPF/pests, drought, salinity and nutrient constraints and the potential role of beneficial actinobacteria in promoting tea crop health. The review also focuses on some of the major PPF associated with tea, such as Exobasidium vexans, Pestalotiopsis theae, Colletotrichum acutatum, and pests (Helopeltis theivora). The phylum actinobacteria own a remarkable place in agriculture due to the biosynthesis of bioactive metabolites that assist plant growth by direct nutrient assimilation, phytohormone production, and by indirect aid in plant defence against PPF and pests. The chemical diversity and bioactive significance of actinobacterial metabolites (antibiotics, siderophore, volatile organic compounds, phytohormones) are valuable in the agro-economy. This review explores the recent history of investigations in the role of actinobacteria and its secondary metabolites as a biocontrol agent and proposes a commercial application in tea cultivation.

Heat shock transcriptional factors (HSFs) are expressed in response to hydrogen peroxide production in grapevines inoculated with Colletotrichum Species

Heat shock transcriptional factors (HSFs) are expressed in plants due to external stress, high temperatures, or pathogenic infections. This study was undertaken to analyze the expression of HSF genes in grapevines inoculated with pathogens. Spores of Colletotrichum acutatum and C. gloeosporioides were inoculated on the leaves, and on immature and mature fruits of ‘Campbell Early’ and creeping grapevine (Vitis flexuosa). The real-time PCR of RNA isolated from infected leaves and fruits showed specific upregulation of HSF11, HSF12, HSF14, and HSF15 subsequent to both high temperature and pathogen infections. However, HSF16 and HSF17 were up-regulated specifically by pathogenic infections only and showed different expression patterns in response to wounding. Among the hydrogen peroxide-related genes, the expression of catalase (CAT) and superoxide dismutase (SOD) was up-regulated by pathogen infections. Concurrently, expression of six HSFs (HSF11, HSF12, HSF14, HSF15, HSF16, and HSF17) dose-dependently increased with accumulation of hydrogen peroxide in the leaves and fruits of grapevines. The expression of selected HSF genes was up-regulated differentially as a defense reaction in ‘Campbell Early’ and V. flexuosa grapevine in response to external stress (such as wounding) and pathogen inoculation. This indicates that expression of some HSFs is regulated through the hydrogen peroxide-mediated pathways in response to pathogens. Further studies determining the mechanism of HSF gene expression induced by external stress are required.

Cross-resistance of Colletotrichum acutatum s. lat. to Strobilurin Fungicides and Inhibitory Effect of Fungicides with Other Mechanisms on C. acutatum s. lat. Resistant to Pyraclostrobin

Colletotrichum acutatum s. lat. 20JDS8 sensitive and 20CDJ6 resistant to pylaclostrobin were used to investigate the cross-resistance with fungicides belonging to the strobilurins and the characteristics of fungicidal controlling activities with different mechanisms against the isolate resistant to the fungicide. The resistant isolate of 20CDJ6 also showed the resistance to azoxystrobin, trifloxystrobin, and kresoxim-methyl, suggesting that there is a cross-resistance relationship. All fungicides with different action mechanisms inhibited mycelial growth of both susceptible and resistant isolates of C. acutatum s. lat., but their disease control effects in fruits were different according to the fungicides. The disease control effect of isopyrazam against 20JDS8 and 20CDJ6 was very low, and fluazinam showed a control effect of 91.9% and 88.1% against 20JDS8 and 20CDJ6 only when it was treated before inoculation by spraying spore suspensions on pepper fruits without wounds. Tebuconazole and prochloraz effectively inhibited not only the mycelial growth of 20JDS8 and 20CDJ6 on potato dextrose agar medium, but also disease incidence in red pepper fruits. As a result of this study, C. acutatum s. lat. 20CDJ6 resistant to pyraclostrobin showed cross-resistance with other strobilurin fungicides. In addition, we think that fluazinam, tebuconazole, and prochloraz can be recommended as alternative fungicides for the control of red-pepper pyranthracnose pathogens resistant pyraclostrobin. However, fluazinam can be effective only if it is treated protectively before the occurrence of the disease.

Orta Anadolu Bölgesinde çilek ekiliş alanlarında görülen fungal hastalıkların tespiti, yaygın olarak yetiştirilen çeşitlerin önemli patojenlere karşı çeşit reaksiyonlarının belirlenmesi

Strawberry diseases, occurring in Central Anatolia region of Turkey was studied in the years 2013-2016 in the strawberry growing areas of Bartın, Kayseri, Konya, and Zonguldak provinces. Totally 515.5 da strawberry fields in the above mentioned provinces were visited and plants showing disease symptoms were counted and their percentages were determined. Foliage and root diseases were determined by using 383 samples collected from various fields. Seventeen fungal pathogens: namely Alternaria alternata (Fr.) Keissler, Boeremia exigua (Desmazières) Aveskamp, Gruyter & Verkley, Mycosphaerella fragariae (Tul.) Lindau., Colletotrichum acutatum J.H. Simmonds, Colletotrichum gleosporoides (Penz.) Penz., Botrytis cinerea (de Bary) Whetzel, Lasiodiplodia theobromae (Pat.) Griffon & Maubl., Diaporthe actinidae N.F. Sommer & Beraha., Diaporthe eres Nitschke, Didymella americana Saccardo ex Saccardo, Didymella pomorum (Thümen) Aveskamp, Gruyter & Verkley, Diplodia seriata de Notaris, Macrophomina phaseolina (Tassi) Goidanich, Peyronellae prosopidis, Phytophthora cactorum (Lebert & Cohn) J.Schröter, Phytophthora plurivora Jung & Burgess., Phytophthora kelmania were identified. A. alternata, was the most frequently isolated fungus and was obtained from 41.94% of the plant samples while root rot diseases caused by various fungi occurred on 38.13% of the fields. Reactions of twelve strawberry cultivars; (Festival, Kabarla, Sweetann, Alison, Osmanlı, Kara çilek, Tüylü, Sabrina, Rubigem, San Andreas, Albion, Festival, Monterey) were tested against the three most widespread pathogen; A. alternata, M. phaseolina and L. theobromae. With the 86% disease severity, Kabarla was found the most susceptible cv. against A. alternata while Tüylü was the most susceptible cv. against M. phaseolina and L. theobromae. Only one variety, Osmanlı, showed low disease intensity against three of the pathogens.

Sterol 14α-Demethylase CaCYP51A and CaCYP51B are Functionally Redundant, but Differentially Regulated in Colletotrichum acutatum: Responsibility for DMI-Fungicide Resistance.

Colletotrichum acutatum, the main pathogen causing anthracnose on chili worldwide, is controlled by tebuconazole [a sterol C14-demethylation inhibitor (DMI) fungicide, abbreviated as Teb] with excellent efficacy. Our previous study exhibited that all C. acutatum isolates were sensitive to Teb while the Colletotrichum gloeosporioides population had developed resistance to Teb on the same fungicide-pressure selection. Therefore, the assessment of Teb-resistance in C. acutatum is impending. Twenty Teb-resistant (TebR) mutants obtained by fungicide domestication and ultraviolet (UV)-mutagenesis displayed similar fitness compared to parental isolates. Data in the current study exhibited that mutations at CaCYP51A and/or overexpression of CaCYP51s were responsible for Teb-resistance. Furthermore, the deletion mutants ΔCaCYP51A and ΔCaCYP51B played different roles in sensitivities to DMIs. Taken together, this study first reported that mutations at CaCYP51A and/or overexpression of CaCYP51s conferred resistance to Teb in C. acutatum, CaCYP51A and CaCYP51B are functionally redundant, but differentially regulated in DMI resistance.

Characterization and necrosis-inducing activity of necrosis- and ethylene-inducing peptide 1-like proteins from Colletotrichum australisinense, the causative agent of rubber tree anthracnose

Colletotrichum australisinense, a member of the Colletotrichum acutatum species complex, is an important pathogen causing rubber tree anthracnose. Genome-wide comparative analysis showed this species complex contains more genes encoding necrosis- and ethylene-inducing peptide 1-like proteins (NLPs) than other Colletotrichum species complexes, but little is known about their necrosis-inducing roles in host. The aim of this study was to analyze NLPs number and type in C. australisinense, and characterize their necrosis-inducing activity in host or non-host. According to phylogenetic relationship, conserved the cysteine residues and the heptapeptide motif (GHRHDWE), 11 NLPs were identified and classified into three types. Five of the eleven NLPs were evaluated for necrosis-inducing activity. CaNLP4 (type 1) could not induce necrosis in host or non-host plants. By contrast, both CaNLP5 and CaNLP9 (type 1) induced necrosis in host and non-host plants, and necrosis-inducing activity was strongest for CaNLP9. CaNLP10 (type 2) and CaNLP11 (type 3) induced necrosis in host but not non-host plants. Substitution of key amino acid residues essential for necrosis induction activity led to loss of CaNLP4 activity. Structural characterization of CaNLP5 and CaNLP9 may explain differences in necrosis-inducing activity. We evaluated the expression of genes coding CaNLP by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qRT-PCR) at different time-points after pathogen infection. It was found that genes encoding CaNLPs with different activities exhibited significantly different expression patterns. The results demonstrate that CaNLPs are functionally and spatially distinct, and may play different but important roles in C. australisinense pathogenesis.

First report of Colletotrichum scovillei causing anthracnose fruit rot on Eriobotrya japonicain in Zhejiang province, China.

Loquat (Eriobotrya japonica (Thunb.) Lindl.) is a fruit tree of high economic impotance in China. In May 2021, fruit rot on cv. "Baozhu" was observed in Yuhang district (119°40'E, 30°09'N), Zhejiang province, at an incidence of more than 30% within five orchards (the total affected area were about 121, 000 m2). Early symptoms of naturally affected fruits were dark brown, necrotic lesion (Fig. 1 A). Lesions subsequently expanded, and orange conidia were observed (Fig. 1 B). Conidia were transferred using a sterilized needle into sterile water, diluted to several conidia (approximately 10 conidia/µL) in the field of light microscope, and spread onto potato dextrose agar (PDA). After 24 h, single colonies were transferred, and six single-spore strain isolated from different fruits or locations were obtained. Six-day old colonies grown on PDA at 27°C had gray-green centers with white edges, and on the reverse side, the centers were brown (Fig. 1 C). Isolate PPGS2 was selected for further characterization. Conidia were unicellular, smooth-walled, hyaline, cylindrical with one rounded and one acute end, or with both ends rounded and they (n = 30) measured 11.1 to 16.0 µm (mean = 13.3 µm) × 3.0 to 4.6 µm (mean = 4.1) (Fig. 1 D). Appressoria were single or in smalln groups, light brown to dark brown, ovoid or elliptical with a smooth or undulate outline, and measured (n = 30) 4.6 to 8.7 µm (mean = 6.5 µm) × 4.0 to 5.6 µm (mean = 4.9 µm) (Fig. 1 H). These features of PPGS2 were consistent with species of the Colletotrichum acutatum species complex (Damm et al. 2012). For species identification, the internal transcribed spacer (ITS), beta-tubulin (TUB), chitin synthase (CHS-1), calmodulin (CAL), and actin (ACT) genes were sequenced (O'Donnell et al. 2000; Weir et al. 2012). The five sequences were deposited in GenBank (OK054581, OK077960, OK077987, OK077988 and OK077989, respectively). A BLAST search of these sequences showed 99% identity with sequences of the ex-holotype C. scovillei CBS126529 (Damm et al. 2012). The evolutionary tree shown that PPGS2 and C. scovillei CBS126529 were clustered in a branch (SH-aLRT/approximate Bayes test/bootstrap support = 88.6/0.999/96). Combining morphological characteristics with phylogenetic analysis, PPGS2 was identified as C. scovillei. To fulfill Koch's postulates, E. japonica fruit were disinfected with 75% alcohol and air-dried for 5 mins to allow the alcohol to volatilize. A conidial suspension (10 mL of 1 × 106 conidia/ml) of PPGS2 was sprayed onto six fruits, and six fruit sprayed with sterilized water served as controls. The experiment was repeated three times. Fruits were kept on a mist bench at 27°C and 80% relative humidity for 5 days. Inoculated fruit developed dark brown necrotic lesions that later fused and expanded (Fig. 1, E-G), whereas control fruit remained symptomless. C. scovillei was re-isolated from lesions of inoculated fruit. C. scovillei has been reported from fruit of Capsicum sp., Musa sp., Mangifera indica, and Clausena lansium in China (Farr and Rossman 2021). To our knowledge, this is the first report of C. scovillei causing anthracnose fruit rot of E. japonica in China. This disease results in severe economic losses both in the field and after harvest, and it is necessary to develop more effective prevention and control strategies.

First report of Colletotrichum fioriniae causing leaf spot on Schima superba in the world.

Schima superba Gardn. et Champ. is a subtropical evergreen tree species naturally distributed mainly in China, Japan, and Vietnam. It is primarily planted for its timber and urban landscaping in China (Ni, 1996). In September 2018, leaves necrotic spots were observed on S. superba in Jiangxi Forest Breeding Center (28°57'19.52" N, 115°39'21.32" E), Jiangxi Province, China. The disease incidence was about 30%. Initially, spots were circular to semicircular, grayish-brown in the center with dark brown margin, then expanded and eventually collapsed into sunken necrotic lesions. To identify the agent, diseased leaves were collected randomly. Pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol (30 s), 3% NaOCl (60 s), and rinsed 3 times in sterile water. These pieces were put on potato dextrose agar (PDA) and cultured at 25 °C. Pure cultures were obtained by monosporic isolation, and 3 isolates (MH-1, MH-2, MH-3) were used for morphological studies and phylogenetic analyses. On PDA, colonies were initially white, cottony, then became pinkish to deep-pink at the center and pink on the reverse. Conidia were fusiform with acute ends, smooth-walled, hyaline, 13.7-18.5 × 4.6-6.1 µm (16.4 ± 1.3× 5.3 ± 0.6 µm, n = 100). Conidiophores were colorless to pale brown, smooth, septate. Conidiogenous cells were colorless to pale brown, smooth, cylindrical to ampulliform. The morphological characteristics fit the descriptions of Colletotrichum acutatum J. H. Simmonds sensu lato (Damm et al., 2012). For accurate identification, genomic DNA of 3 isolates was extracted, and the internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin 2 (TUB2), and chitin synthase (CHS-1) were amplified and sequenced using the corresponding primers (Weir et al., 2012). The sequences were deposited in GenBank (ITS: MZ325946, MZ325947, MW584318; ACT: MZ399375, MZ419566, MW661171; CHS-1: MZ399376, MZ419567, MW661172; MZ399377, GAPDH: MZ419568, MW661173; TUB2: MZ399378, MZ419569, MW661174). Five loci were concatenated, and the aligned sequences (1528bp) were 99.89% homologous to ex-type C. fioriniae (Marcelino & Gouli) R. G. Shivas & Y. P. Tan CBS128517. Phylogenetic analysis using the maximum likelihood showed that 3 isolates were clustered in C. fioriniae clade with 100% bootstrap support. Based on the multi-locus phylogeny and morphology, 3 isolates were identified as C. fioriniae. Pathogenicity tests were performed on 36 seedlings of S. superba (2-year-old). The leaves were wounded slightly and inoculated with a drop of spore suspension (106 conidia/mL). The sterile water was used as controls. All the tested leaves were covered with black plastic bags to keep them moist for 2 days. All seedlings were placed in the greenhouse (25 °C, 12 h light/dark) for 10 days, and all inoculated leaves had typical symptoms. The controls were asymptomatic. The same fungus was reisolated from the lesions, fulfilling Koch's postulates. Colletotrichum fioriniae was described as a new species from the C. acutatum s. l. (Shivas et al., 2009), and it was an important plant pathogen, such as Pyrus spp. (Pavlović et al., 2019), Morus alba L. (Xue et al., 2019), and so on. This is the first report of the newly emerging disease of S. superba caused by C. fioriniae in the world, and its potential threat should be evaluated in the future. This study provided crucial information for epidemiologic studies and appropriate control strategies.

Ontogenic Susceptibility of Grapevine Clusters to Ripe Rot, Caused by the Colletotrichum acutatum and C. gloeosporioides Species Complexes.

Natural infection by Colletotrichum spp. and the subsequent development of ripe rot were observed in susceptible grape (Vitis vinifera) clusters either protected or exposed to environmental conditions and naturally occurring inoculum by the application or removal of paper bags at various phenological stages at two Mid-Atlantic vineyards. During each of the three experimental seasons, most treatments with grape clusters that were exposed between veraison and harvest developed significantly more severe ripe rot than clusters that were exposed during only the early season or protected throughout the entire season. Spore traps were placed in one vineyard over two seasons and were analyzed via quantitative PCR. DNA of the Colletotrichum acutatum and C. gloeosporioides species complexes was detected from the bloom to the harvest stage, with a higher quantity of C. acutatum DNA than C. gloeosporioides DNA. From ripe rot symptomatic clusters, 417 isolates were collected, and a multilocus phylogenetic analysis of 51 representative isolates identified six Colletotrichum spp., with C. fioriniae (C. acutatum complex) being the most frequently isolated. Weather data were also monitored, and ripe rot-conducive conditions were observed at multiple times throughout each season. In summary, only clusters that were exposed to inoculum and environmental conditions in the late season developed severe ripe rot. The data collected in this study suggest that grape clusters have ontogenic susceptibility to ripe rot, becoming more susceptible as they mature, in contrast to the susceptible bloom stage for other fruit rotting diseases of grapevine.

From Glaciers to Refrigerators: the Population Genomics and Biocontrol Potential of the Black Yeast Aureobasidium subglaciale.

ABSTRACTApples are affected by numerous fungi known as storage rots, which cause significant losses before and after harvest. Concerns about increasing antimicrobial resistance, bans on various fungicides, and changing consumer preferences are motivating the search for safer means to prevent fruit rot. The use of antagonistic microbes has been shown to be an efficient and environmentally friendly alternative to conventional phytopharmaceuticals. Here, we investigate the potential of Aureobasidium subglaciale for postharvest rot control. We tested the antagonistic activity of 9 strains of A. subglaciale and 7 closely related strains against relevant phytopathogenic fungi under conditions simulating low-temperature storage: Botrytis cinerea, Penicillium expansum, and Colletotrichum acutatum. We also investigated a selection of phenotypic traits of all strains and sequenced their whole genomes. The tested strains significantly reduced postharvest rot of apples at low temperatures caused by B. cinerea, C. acutatum (over 60%), and P. expansum (about 40%). Several phenotypic traits were observed that may contribute to this biocontrol capacity: growth at low temperatures, tolerance to high temperatures and elevated solute concentrations, and strong production of several extracellular enzymes and siderophores. Population genomics revealed that 7 of the 15 strains originally identified as A. subglaciale most likely belong to other, possibly undescribed species of the same genus. In addition, the population structure and linkage disequilibrium of the species suggest that A. subglaciale is strictly clonal and therefore particularly well suited for use in biocontrol. Overall, these data suggest substantial biological control potential for A. subglaciale, which represents another promising biological agent for disease control in fresh fruit.IMPORTANCE After harvest, fruits are often stored at low temperatures to prolong their life. However, despite the low temperatures, much of the fruit is lost to rot caused by a variety of fungi, resulting in major economic losses and food safety risks. An increasingly important environmentally friendly alternative to conventional methods of mitigating the effects of plant diseases is the use of microorganisms that act similarly to probiotics—occupying the available space, producing antimicrobial compounds, and consuming the nutrients needed by the rot-causing species. To find a new microorganism for biological control that is particularly suitable for cold storage of fruit, we tested different isolates of the cold-loving yeast Aureobasidium subglaciale and studied their phenotypic characteristics and genomes. We demonstrated that A. subglaciale can significantly reduce rotting of apples caused by three rot-causing molds at low temperatures and thus has great potential for preventing fruit rot during cold storage.

Sensitivity of Colletotrichum acutatum Species Complex from Strawberry to Fungicide Alternatives to Quinone-Outside Inhibitors.

Colletotrichum acutatum is a species complex that causes anthracnose fruit rot and root necrosis on strawberry. The major and minor species within the complex that affect strawberry production are C. nymphaeae and C. fioriniae, respectively. The disease can significantly reduce yield under conducive weather, and its management has greatly relied on quinone-outside inhibitor fungicides (QoI). However, due to the emergence of resistant isolates, such products are no longer effective. Therefore, alternative fungicides were investigated. C. nymphaeae and C. fioriniae isolates were collected from multiple strawberry fields in the United States from 1995 to 2017. The sensitivity of benzovindiflupyr, penthiopyrad, pydiflumetofen, fluazinam, fludioxonil, and cyprodinil was assessed by invitro and invivo assays. Both Colletotrichum species were sensitive to benzovindiflupyr, penthiopyrad, fluazinam, and fludioxonil based on mycelial growth assays. Interestingly, of these products, only penthiopyrad did not inhibit conidial germination at 100 µg/ml. For cyprodinil, C. nymphaeae was sensitive based on the mycelial growth, whereas C. fioriniae was not inhibited. Neither species was inhibited by pydiflumetofen in mycelial growth, conidial germination, nor detached fruit assays. The prepackaged mixtures fludioxonil + cyprodinil and fludioxonil + pydiflumetofen were effective in a field trial; however, their use should be carefully considered because of the lack of efficacy of one of the compounds in the mixture. This study sheds light on the potential registration of products alternative to QoIs, such as benzovindiflupyr and fluazinam, which could improve the management of strawberry anthracnose.

Antifungal effects of Paeonia lactiflora seed extracts

Paeonia lactiflora is a plant resources widely distributed in Asia. The root of Paeonia lactiflora have been used to traditional herbal medicine for blood stasis syndrome in Korea. Until now, many studies have been conducted on the root of Paeonia lactiflora, but studies on seed are insufficient. This study was carried out to determine the inhibitory effect of Paeonia lactiflora seed extract against the phytopathogenic fungi Alternaria panax, Botrytis cinerea, Colletotrichum acutatum, Cylindrocarpon destructans, Colletotrichum gloseosporiades, Phytophthora cactorum, and Pythium ultimum. The results demonstrated that the seed extract showed potent antifungal effects against Alternaria panax, Colletotrichum acutatum, Cylindrocarpon destructans, Colletotrichum gloseosporiades, Phytophthora cactorum, and Pythium ultimum. Especially, the Paeonia lactiflora seed extract showed the highest antifungal activity against Pythium ultimum and Phytophthora cactorum, and the antifungal activity was not affected by heat treatment. Therefore, the antifungal component of the Paeonia lactiflora seed extract was concluded thermostable and it is suggested that the Paeonia lacflora seed extract has stability for antifungal activity maintaining. These results suggested that Paeonia lacflora seed extract may be useful as a natural antifungal agent to prevent plant diseases.

Induction of defense mechanisms in avocado using Mexican oregano oil-based antifungal sachet

Active sachets can be used to manage postharvest losses associated with phytopathogens in fruit and vegetables. Diseases associated with phytopathogens are the principal causes of avocado losses postharvest. This study was performed to develop antifungal active sachet-based oregano oil microencapsulated with starch/agave fructans that allows in vivo and in vitro control of phytopathogens associated with avocado decay. In addition, avocado-sachet interactions were studied. Oregano oil sachets inhibited 100% of the in vitro growth of Colletotrichum gloeosporioides, Colletotrichum acutatum, Diaporthe passiflorae, and Neoscytalidium hyalinum at 30 °C for 12 d The efficacy of the oregano oil sachets was confirmed on avocados inoculated with C. gloeosporioides. Active sachets reduced the injury area of anthracnose infection in avocado without negative effects on the color or firmness of the fruit, compared to untreated control. Treatment also caused significantly higher (p < 0.05) phenylalanine ammonia-lyase, chitinase, β-1,3-glucanase, catalase and peroxidase activities compared to the control fruit. In addition, antifungal sachets significantly enhanced the contents of total flavonoids and phenolic compounds in treated avocados.

Inhibitory Effect of Syzygium aromaticum L. Essential Oil Against the Fungal Pathogens of Capsicum annuum L.

Fungal pathogens are causative agent of pepper diseases that affect the yield loss. At least 10% of food loss in the developing country caused by plant diseases. Clove oil (Syzygium aromaticum L.) was reported to be able to control pathogenic fungi. In order to evaluate the effect of clove oil in inhibiting the growth of the pepper disease, three types of major causal agent of pepper diseases were used namely Colletotrichum acutatum, Phythopthora capsici, and Pythium sp. The experiment was evaluated in vitro. The three pathogens were treated with the essential oil in different concentration according to IC50 result on each fungi. The clove oil showed inhibitory effect against tested fungi. The Syzygium aromaticum L. essensial oil showed the best inhibitory effect at concentration 340 µl/L, 180 µl/L and 100 µl/L for C. acutatum, P. capsici, and Pythium sp. respectively.

Development of a nested PCR assay for detecting Colletotrichum siamense and Colletotrichum fructicola on symptomless strawberry plants.

Anthracnose is a major disease of strawberry that seriously impacts the strawberry industry. To prevent the spread of anthracnose through symptomless plants, it is important to detect pathogenic Colletotrichum spp. at the latent infection stage in the nursery. Previous PCR-based methods developed for the diagnosis or detection of Colletotrichum acutatum and Colletotrichum gloeosporioides have used primers targeting the internal transcribed spacer region of ribosomal DNA, β-tubulin gene, or mating type gene. In this study, to specifically detect Colletotrichum siamense and Colletotrichum fructicola, the most predominant and virulent Colletotrichum species causing strawberry anthracnose in Taiwan, we conducted a comparative genomics analysis of 29 Colletotrichum spp. and identified a non-conserved 1157-bp intergenic region suitable for designing specific primers for a nested PCR assay. In silico analysis and actual tests suggested that the new nested PCR assay could detect pathogenic C. siamense and C. fructicola, but not other strawberry pathogens (Botrytis sp., Fusarium spp., Neopestalotiopsis rosae, and Phytophthora sp.) or ubiquitous saprophytes (Fusarium spp. and Trichoderma spp.). The inner to outer primer ratio was optimized to 1:10 to eliminate unexpected bands and enhance the signal. The assay could detect as little as 1 pg of C. siamense genomic DNA, which corresponds to ~15 cells. Application of the new detection assay on 747 leaf samples collected from 18 strawberry nurseries in 2019 and 2020 showed that an average of 20% of strawberry mother plants in Taiwan were latently infected by C. siamense or C. fructicola. The newly developed assay is being applied to facilitate the production of healthy strawberry runner plants in Taiwan.

Comparison and Characterization of Colletotrichum acutatum the Main Causative Agent of Colletotrichum Leaf Disease (CLD) of Rubber (Hevea brasiliensis) with Newly Identified Causative Agent Colletotrichum siamense

Colletotrichum acutatum has been identified as the major pathogenic species of CLD in rubber. Recently, C. siamense was identified as another causative agent of this disease from Sri Lankan rubber plantations. The present study was focused to compare C. acutatum and C. siamense giving special reference to cultural and reproductive characteristics, fungicide sensitivity and pathogenicity. Comparison of new pathogens with already existing pathogens are required for understanding of new pathogen population. That knowledge will be important to improve management strategies, quarantine regulations and to protect crop avoiding a huge economical lose. C. siamense was isolated from symptomatic rubber leaves on to Potato Dextrose Agar (PDA) and C. acutatum was collected from department culture collection. Cultural characters were observed after 10 days of incubation. Reproductive characters were compared using slide culture technique. Conidia concentrations were detected using haemocytometer. Growth rates were measured along two perpendicular axis on PDA plates. Fungicide sensitivity was tested with 0.01-500 ppm carbendazim following poisoned food technique. Pathogenicity test was performed using 5×105 conidia/ml suspension on detached rubber leaves and Disease Index (DI) was calculated. Colonies were circular, smooth and cloudy with raised elevation, opaque nature and entire margin. Both upper and lower surfaces of C. siamense were light grey with orange conidiomata on the lower surface. C. acutatum produced white to light grey colonies which were initially salmon coloured in lower surface. Conidia in both isolates were aseptate one celled, hyaline, smooth-walled and granular with size of 14-16 μm×4-5 μm. C. siamense produced cylindrical, both ends rounded spores with 4×105 conidia/ml concentration while C. acutatum produced fusiform conidia with 2.08×107 conidia/ml upon 7 days of incubation. However, the growth of C. siamense in PDA was higher (1.60 cm/day) than the growth of C. acutatum (1.48 cm/day). Although C. siamense inhibited 100% of its growth even at 0.5 ppm of carbendazim, C. acutatum was extremely tolerant to carbendazim and couldn’t inhibit the growth even at 500 ppm. In the pathogenicity test, lesions were produced at all inoculation points but the size and type of lesions produced by both isolates were different. C. acutatum produced darker anthracnose-like watery lesions with 3.00 DI value and C. siamense produced slightly dark watery lesions with DI value of 2.00. According to the characters of both isolates, C. acutatum can be identified as a severe pathogen than C. siamense.

Short Communication: Effectiveness of cinnamon leaf extract to control anthracnose disease on large chilies in Bali, Indonesia

Abstract. Darmadi AAK, Suriani NL, Ginantra IK, Sudirga SK. 2022. Short Communication: Effectiveness of cinnamon leaf extract to control anthracnose disease on large chilies in Bali, Indonesia. Biodiversitas 23: 2859-2864. The use of synthetic fungicides for plants is very dangerous because it has a negative impact on the environment. In the context of implementing eco-friendly, sustainable agriculture, efforts need to be made to reduce the negative impact caused by the use of synthetic fungicides. This study aimed to test the effectiveness of a crude extract of cinnamon leaves, observe the inhibition effect on fungi by scanning electron microscope, and test the formulation of cinnamon leaf extract against anthracnose disease on large chilies in the field. Cinnamon leaf crude extract showed inhibition zone of 35 mm diameter against Colletotrichum acutatum. The extract showed strong inhibition so further used as biofungicide in field experiments. Based on scanning electron microscopy, cinnamon leaf extract can damage to the fungal cell wall. Cinnamon leaf extract formula was found effective in reducing disease percentage and intensity of anthracnose disease in experimental fields. In the treatment, a concentration of 0.5% to 1.5% can inhibit the percentage of disease ranging from 29.4% to 100%. Concentrations of 0.5% to 1.5% can prevent disease severity from 35.8% to 100%.

First Report of Anthracnose Caused by Colletotrichum acutatum on Olive Fruits in Pakistan.

Anthracnose symptoms on olive (Olea europaea) fruits cv. "Gamlick" were found in farmer orchards in Chakwal, Punjab, Pakistan (32° N and 72° E), with an average prevalence of 59%. Fruit symptoms start as thin, black, sunken lesions with a watery appearance that grow in diameter and coalesce into a large sunken soft zone. Lesions on mature fruit become noticeable in 5 to 6 days after infection, if temperatures are favorable (28°C). On the fruit lesion, orange conidial masses in dispersed or concentric circle arrangement can appear. Fragments (5 mm) were taken from the margins of fruit lesions and surface-sterilized with 70% ethanol (1 min) and 1% NaClO (2 min), cleaned with sterile purified water, blotted dry, and plated on potato dextrose agar (PDA) in Petri dishes. The petri plates were incubated at 27°C. A fungus was consistently isolated, and thirty-five isolates were characterized. Aerial mycelia from olive isolates Colonies were compact, initially white or cream white, then grey, and eventually dark grey, with conidium masses forming in the middle. Mycelium is branched, septate, and hyaline. Conidia are hyaline, aseptate, fusiform, or often cylindrical, with obtuse apices and tapering bases. Their mean size was 8.5µm in length and 3.0 µm in width. Based on morphological features, the fungus was tentatively identified as Colletotrichum acutatum (Agosteo G.E., 2010). The identification was confirmed by amplification and sequencing of a representative isolate's internal transcribed region (ITS), Beta- tubulin region (TUB2), Actin region (ACT), and Glyceraldehyde 3-phosphate dehydrogenase region (GAPDH) with the primers ITS1/ITS4 (Gardes & Bruns 1993), TUB4/TUB5 (Woudenberg et al. 2009), ACT1/ACT3 (Carbone & Kohn 1999) and GDF1/GDR1 (Guerber et al. 2003). BLAST analysis revealed 100% identity for ITS, GAPDH and ACT and 99% identity for TUB, between the sequences of the olive fruit isolate (GenBank Accessions MW647502, MZ436968, MZ714412 and MW810331, respectively) and sequences of C. acutatum reference isolates (GenBank Accessions GO613492, KF975660.1, MT274752.1 and MH547616 respectively). Phylogenetic analysis based on ITS, GAPDH and TUB regions of the olive fruit isolates and reference isolates of various Colletotrichum species using the MEGA X software program confirmed the isolate from olive was C. acutatum. The fungal isolate was deposited as a living culture in the Barani Agricultural Research Institute's fungal culture collection center (BACA.9381). Pathogenicity tests were conducted with this isolate by placing a 20 µl drop of a conidial suspension (3 × 107conidia ml-1) on five healthy olive cv. Gemlik fruits. As a control, five non-inoculated olive fruits were used. Fruits were placed at a temperature of 27°C with artificial light and a photoperiod of 12 hours. Anthracnose symptoms developed only on inoculated fruits after seven days of inoculation. The fungus was re-isolated from symptomatic fruits, and its identity was confirmed through morphological characteristics, thus verifying Koch's postulates. To the best of our knowledge, this is the first report of C. acutatum infecting olive fruits in Chakwal region of Pakistan.