Colletotrichum Acutatum Research Articles

Olive-tree physiological response to biotic and abiotic stress fruit yield, oil quality and tolerance to Anthracnose: Doctoral thesis abstract

The olive tree is highly tolerant to water deficit and in humid climate regions, such as Uruguay, the need to irrigation is questioned. However, Uruguay's climate is characterized by high interannual variability, resulting in periods of water deficit. In the event of a water deficit, plants trigger a series of defense mechanisms to maintain cellular homeostasis, including increased antioxidant enzyme activity and cuticle thickening to prevent transpiration. These responses are common during abiotic stress and biotic stress caused by pathogen infections. Anthracnose, caused by the Colletotrichum complex, is a major disease in olive trees, leading to reduced fruit yield and oil quality. Mild drought stress has been found to activate the basal defense response, enhancing plant tolerance to pathogen infections. Therefore, the hypothesis was that exposing olive plants to moderate water deficit during the lipogenesis stage would trigger the basal defense response, increasing fruit tolerance to biotic stress caused by Colletotrichum acutatum. The study also examined whether this response was sustained under moderate water deficit. Results showed that water deficit induced anatomical and biochemical changes in olive fruits, promoting anthracnose tolerance with reduced incidence and severity of C. acutatum infection both in vitro and in vivo. Fruit weight and pulp/pit ratio decreased in response to water deficit, while cuticle thickness and fruit polyphenol content increased. An induction of enzymes related to hydrogen peroxide scavenging were observed, in particular PER and CAT. This enzymatic activity was induced by the pathogen inoculation treatment and was higher in the non-irrigated treatment. Severe water deficit resulted in significant reductions in fruit weight and oil content, but the fruits exhibited enhanced tolerance to anthracnose infection.

In vitro Antifungal Efficacy of Some Plant Extracts against Fungal Pathogens Causing Diseases in Solanaceous Crops

Background: This research aimed to assess the antifungal potential of oil and organic leaf extracts obtained from Ocimum gratissimum, Kopsia fruticosa and Hibiscus rosa-sinensis against fungal pathogens affecting solanaceous crops. Methods: Oil of O. gratissimum was obtained by hydro-distilled method using Clevenger apparatus. Leaf extracts of K. fruticosa and H. rosa-sinensis were extracted using three organic solvents namely hexane, ethanol and methanol. The antifungal activity of the oil and organic leaf extracts against the test fungal pathogens was determined by the agar well diffusion method. The efficacy of the oil and organic leaf extracts against the pathogens was evaluated by their minimum inhibitory concentration (MIC), ability to inhibit spore germination and growth kinetic assay. Result: The oil of O. gratissimum (1000 ppm) significantly inhibited the growth of Cercospora capsici (81.4%), Alternaria alternata (73.7%) and Fusarium oxysporum (68.7%). Hexane extract of K. fruticosa and methanol extract of H. rosa-sinensis (1500 µg/well) displayed strong antifungal activity against Colletotrichum acutatum with inhibition percentages of 75.9% and 82.4% respectively. Similarly, ethanol extract of K. fruticosa and methanol extract of H. rosa-sinensis showed effective inhibition against C. capsici and A. alternata. The oil of O. gratissimum showed MIC values of 50, 100 and 200 µg/mL against C. capsici, A. alternata and F. oxysporum respectively. Ethanol extract of K. fruticosa showed MIC values ranging from 100 to 350 µg/mL, while H. rosa-sinensis methanol extract displayed MIC values ranging from 50 to 200 µg/mL against the fungal pathogens. The oil extract also demonstrated strong inhibition of spore germination of the test fungi. Our study suggests the potential of plant-derived essential oils and extracts as effective antifungal agents for crop protection.

Antifungal Activity of Culture Filtrate from Endophytic Fungus Nectria balsamea E282 and Its Fractions against Dryadomyces quercus-mongolicae

A key role that fungal endophytes play in interacting with their host plant can be defined by the fact that they promote the growth of plants and enhance the tolerance of the host against plant pathogens using bioactive compounds that they produce. Several studies utilizing endophytic fungi as a source of biological control against plant pathogens were conducted, and a representative example includes Aureobasidium protae from common wheat (Triticum aestivum), which inhibited the mycelial growth of Fusarium graminearum and Fusarium culmorum, causal agents of Fusarium head blight disease. Our previous study indicated that three endophytic fungal isolates, E089 (Daldinia childiae), E282 (Nectria balsamea), and E409 (Colletotrichum acutatum), showed antifungal activities against D. quercus-mongolicae, an ascomycetous fungus that is reported to be associated with oak mortality in South Korea. The objectives of this study were to optimize and evaluate antifungal efficiency for these endophytic fungi against D. quercus-mongolicae, and this was achieved using culture filtrate retrieved from the three above-mentioned endophytes and fractions isolated from the culture filtrate. Of those, the culture filtrate from E282 showed higher mycelial growth and sporulation inhibitions on PDA medium where D. quercus-mongolicae was grown. In addition, three fractions, including hexane, CHCl3, Et2O, and H2O, were tested for antifungal activities against D. quercus-mongolicae. The results revealed that the Et2O fraction showed higher mycelial growth and sporulation inhibition rates. Taking these results together, the endophytic fungus, N. balsamea, which exhibited high antifungal efficiency, can be effectively used as a biocontrol agent for the management of oak wilt disease in the country.

Phytoconstituents and Ergosterol Biosynthesis-Targeting Antimicrobial Activity of Nutmeg (Myristica fragans Houtt.) against Phytopathogens.

In recent years, nutmeg (Myristica fragans Houtt.) has attracted considerable attention in the field of phytochemistry due to its diverse array of bioactive compounds. However, the potential application of nutmeg as a biorational for crop protection has been insufficiently explored. This study investigated the constituents of a nutmeg hydroethanolic extract via gas chromatography-mass spectrometry and vibrational spectroscopy. The research explored the extract's activity against phytopathogenic fungi and oomycetes, elucidating its mechanism of action. The phytochemical profile revealed fatty acids (including tetradecanoic acid, 9-octadecenoic acid, n-hexadecanoic acid, dodecanoic acid, and octadecanoic acid), methoxyeugenol, and elemicin as the main constituents. Previously unreported phytochemicals included veratone, gelsevirine, and montanine. Significant radial growth inhibition of mycelia was observed against Botrytis cinerea, Colletotrichum acutatum, Diplodia corticola, Phytophthora cinnamomi, and especially against Fusarium culmorum. Mode of action investigation, involving Saccharomyces cerevisiae labeled positively with propidium iodide, and a mutant strain affected in ERG6, encoding sterol C-24 methyltransferase, suggested that the extract induces a necrotic type of death and targets ergosterol biosynthesis. The evidence presented underscores the potential of nutmeg as a source of new antimicrobial agents, showing particular promise against F. culmorum.

Identification of <i>Rca2</i> and <i>Rpf1</i> resistance alleles in strawberry breeding forms of intervarietal hybrid origin

Background. The creation of varieties with genetic resistance to fungal pathogens is one of the most important directions in strawberry breeding. A promising direction of accelerated selection of valuable specimens is molecular screening for the presence in their genotype of diagnostic DNA markers, linked to target genes of agrobiological traits. Objectives of the work were determination of the allelic state of the Rca2 (resistance to anthracnose) and Rpf1 (resistance to red stele root rot) genes in selected strawberry forms to identify genotypes resistant to Colletotrichum acutatum Simmonds and Phytophthora fragariae var. fragariae Hickman.Material and methods. The object of research was breeding forms of strawberries of intervarietal hybrid origin. As a result of 22 crossing combinations, 61 selection forms were obtained, in which the allelic composition of resistance genes was studied. The Rca2 anthracnose resistance allele was identified by means of the STS-Rca2_240 marker. The Rpf1 red stele root rot resistance allele was identified with the help of SCAR-R1A marker. Results. At least one of the two genes was identified in 20 selected strawberry forms (32.8%). The STS-Rca2_240 marker was identified in 15 strawberry seedlings (22.9%) from six hybrid combinations ‘Vicoda’ × ‘Roxana’, ‘Florence’ × ‘Faith’, ‘Asia’ × ‘Aprica’, ‘San Andreas’ × ‘Monterey’, ‘Alisa’ × ‘Quicky’, ‘Quicky’ × ‘Olympia’). The SCAR-R1A marker was identified in five strawberry seedlings (8.2%) from four hybrid combinations (‘Privlekatelnaya’ × ‘Bylinnaya’, ‘Feyerverk’ × ‘Bylinnaya’, ‘Bylinnaya’ × ‘Feyerverk’, ‘Bylinnaya’ × ‘Olimpiyskaya nadezhda’). It has been established that all strawberry selected forms with identified marker fragments for resistance genes are characterized by a heterozygous combination of alleles.Conclusions. Promising sources of resistance alleles are strawberry selected forms: 2/1-24, 2/1-34 (‘Quicky’ × ‘Olympia’), 3/9-5 (‘Florence’ × ‘Faith’), 3/12-2, 3/12- 9 (‘Alisa’ × ‘Quicky’), 4/7-10, 4/7-19, 4/7-20 (‘Asia’ × ‘Aprica’), 5/2-26, 5/2-32 (‘San Andreas’ × ‘Monterey’), 9/ 1-11, 9/1-12, 9/1-13, 9/1-32, 9/1-37 ‘Vicoda’ × ‘Roxana’) in case of Rca2 gene; 72-24, 72-71 (Privlekatelnaya × Bylinnaya), 69-29 (‘Feyerverk’ × ‘Bylinnaya’), 62-41 (‘Bylinnaya’ × ‘Feyerverk’), 61-15 (‘Bylinnaya’ × ‘Olimpiyskaya nadezhda’) in case of Rpf1 gene.

In silico design of sgRNA for CRISPR/Cas9-mediated FaRALF33 gene mutagenesis to decrease the infection process to Colletotrichum acutatum in strawberry

Rapid alkalinization factors (RALFs) are ubiquitous cysteine-rich peptides present in plants. They exert diverse functions as hormonal signals in various processes, including cell growth, root elongation, and fertilization. RALF peptides can also act as negative regulators of the plant immune response, inhibiting the formation of the signal receptor complex for immune activation. In Fragaria × ananassa, silencing of FaRALF33 gene plays a key role in the defense against the fungal pathogen Colletotrichum acutatum. In this study, single-guide RNA (sgRNAs) were designed in silico for clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) 9-mediated FaRALF33 gene mutagenesis in F. × ananassa for the reduction of C. acutatum infection. FaRALF33 was compared with homologous RALF33 sequences from other plant species, showing that the amino acid sequence of FaRALF33 presents typical sequences of known RALF peptides in RRILA proteolytic site, in addition to tight clustering presented by FaRALF33 with FvRALF33. The online tool CHOPCHOP provided 73 hits for FaRALF33 gene, selecting two sgRNA sequences for mutagenesis, sgRNA 1 (5’-CGACTCTCCCATCTCTTGGACT-3’) and sgRNA 2 (5’-GCAAGCAACGGCAGCGATCA-3’). The predicted secondary structures of the selected sgRNAs presented efficient structures in targeted mutagenesis. The pCas9-TPC-GFP-2XsgRNA vector for CRISPR/Cas9-mediated FaRALF33 gene mutagenesis was designed in silico with two sgRNA sequences (with Arabidopsis thaliana U6-26 promoters) and a green fluorescent protein marker.

Vitis species and varietal resistance of mature berries against inoculation of an isolate of the Colletotrichum acutatum complex that causes grape ripe rot

<i>Vitis</i> species and varietal resistance of mature berries against inoculation of an isolate of the <i>Colletotrichum acutatum</i> complex that causes grape ripe rot

First Report of Anthracnose Caused by Colletotrichum nymphaeae on Strawberry Fruits in Central Argentina.

Strawberry (Fragaria x ananassa) production in Argentina extends to around 1700 hectares. Coronda city, located in Santa Fe province, is an important strawberry producer due to ideal agroecological conditions for culture and a high specialization for production. In November 2021, anthracnose symptoms were observed on strawberries cvs. 'San Andreas' and 'Splendor' in Coronda (31°58'S, 60°55'W), central Argentina. During these years, the incidence of the disease reached 40% of the production. Symptoms included 2-3 mm circular to irregular dark brown spots which enlarged rapidly and became sunken. Under high humidity conditions, concentric rings of pinhead-size salmon-colored acervuli developed on the lesions. The causal agent was isolated by touching acervuli with a sterile needle and monosporic cultures were obtained on PDA after 10 days at 25°C, with a 12-h light period. Colonies were white to gray on the top and orange on the underside, where concentric rings of salmon acervuli were clearly distinguished. The width and length of one hundred conidia were examined in three isolates (CF1, CF2, and CF3), ranging from 3.27 to 5.53 μm (avg.= 4.3 μm), and from 10.37 to 19.52 μm (avg.= 14.27 μm), respectively. The conidia were hyaline, smooth-walled, aseptate, and cylindric-clavate with one end round and one end acute. These morphological characteristics correspond to species belonging to the C. acutatum complex (Damm et al. 2012; Liu et al. 2022). To accurately identify the species, DNA was extracted from isolates, and ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and histone (HIS3) genes were partially amplified and sequenced (Vieira et al. 2020). TUB2, GAPDH, and HIS3 sequences presented a 100% of identity with species of Colletotrichum nymphaeae. The nucleotide sequences were deposited in GenBank (OR271556-OR271558, TUB2; OR271559-OR271561, GAPDH; and OR271562-OR271564, HIS3). Multilocus phylogenetic analyses performed with reference sequences (Damm et al. 2012) showed that the three isolates clustered with C. nymphaeae, in accordance with BLAST results. To confirm pathogenicity, each isolate was inoculated in eight detached fruits of the cultivar from which it was originally obtained. Two drops of 10 μl of conidial suspension (1x105 conidia per ml) were deposited in non-wounded areas on fruits previously disinfested with 1% sodium hypochlorite solution for 1 min and rinsed twice with sterile distilled water. Drops of sterile water were deposited in eight fruits as control. Pathogenicity tests were repeated twice. Fruits were kept in moist chamber (80+5% relative humidity) at 25°C for ten days. First symptoms appeared 4 days after inoculation. After that, all of the isolates produced symptoms identical to those previously described, whereas the controls remain symptomless. The pathogen was re-isolated from lesions, and identified as C. nymphaeae by morphological characteristics and based on the TUB2 sequences, as previously described. Strawberry anthracnose in Argentina was previously associated with Colletotrichum acutatum, C. gloeosporioides and C. fragariae species based on morphological characteristics (Ramallo et al. 2000; Monaco et al. 2000) but molecular identification was not performed until today. To our knowledge, this is the first report of C. nymphaeae causing anthracnose on strawberry in Argentina. This accurate identification will help to develop more efficient management strategies.

First report of Colletotrichum grossum causing apple bitter rot worldwide (Italy).

Apple bitter rot is a globally widespread disease that is observed on fruits both pre-harvest and post-harvest, contributing to considerable economic losses. While the Colletotrichum acutatum species complex are predominant in Europe (Baroncelli et al. 2014; Amaral Carneiro and Baric 2021), in recent years, the Colletotrichum gloeosporioides species complex are emerging, raising many concerns (Amaral Carneiro et al. 2023). Circular, slightly sunken, brown lesions with acervuli produced in concentric spots were observed on 'Story® Inored' cultivar harvested in September 2022 from an organic orchard in Masi (Padova province, Italy), with a disease incidence close to 30%. From ten diseased apples, tissue samples were excised under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to potato dextrose agar medium and incubated in the dark at 25 °C for 7 days, whereafter five single-spore cultures were obtained. Pure colonies grown at 25 °C for 7 days appeared light gray-white on the upper side with floccose aerial mycelium, while the reverse side was dark gray with a distinct margin. Conidia were hyaline, cylindrical in shape with both ends rounded or one end acute and measured 16.6 ± 1.4 × 6.1 ± 0.5 μm [mean ± SD] (n=50) as described by Diao et al. 2017. To identify the species, genomic DNA of a representative isolate (C38) was extracted, beta-tubulin (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), Apn2-Mat1-2 intergenic spacer (ApnMat) genes and the internal transcribed spacer (ITS) region, were amplified by PCR and Sanger sequenced (Rojas et al. 2010; Weir et al. 2012). The obtained DNA sequences of, TUB2, CAL, GAPDH, GS, ApnMat and ITS were submitted to GenBank under the accession numbers OR025589, OR025586, OR025587, OR025588, OR025585 and OR004800, respectively. A MegaBLAST analysis resulted 100 % identity to the epitype CAUG7 of Colletotrichum grossum (Diao et al. 2017) for GAPDH (KP890159), for TUB2 (KP890171), 99.85% for CAL (KP890147) and 99,5 % for ITS (KP890165). The phylogenetic tree constructed by concatenation with the obtained sequences, as well as references, revealed that the C38 isolate clustered within C. grossum, confirming the BLAST approach. Pathogenicity tests were performed on 40 'Story® Inored' apples cleaned and wounded with a sterilized needle and exposed to two different conditions: 20 apples (10 inoculated with 20 μl of spore suspension (105 ml-1) and 10 with sterile water as control) were incubated at 20°C with a 12-hour photoperiod for 14 days, while the remaining 20 apples, prepared with same approach, were placed at 1°C for 3 months, then at room temperature for 14 days. Symptoms appeared after 6 days on apples incubated at 20°C, whereas those stored at 1°C displayed symptoms at 11 days after being placed at room temperature. In both conditions, lesions were similar to those observed on the original fruits; while the controls remained asymptomatic. Identity of reisolated fungal colonies was confirmed by CAL, GAPDH and GS region sequence analysis. C. grossum has been reported rarely: in 2017 on Capsicum annuum var. grossum in China, in 2018 on Mangifera indica leaves in Cuba, and in 2021 on Rhyncospermum jasminoides in Italy (Diao et al. 2017; Manzano León et al. 2018; Guarnaccia et al. 2021). To the best of our knowledge, this is the first report of apple bitter rot caused by Colletotrichum grossum worldwide.

Lupinus mutabilis Breeding in the Andes of Ecuador, Peru, and Bolivia: A Review

Lupinus mutabilis, also known as tarwi or chocho, is an important agricultural species that has been cultivated in South America since ancient times. Tarwi is native to the Andean regions of Peru, Bolivia, and Ecuador and has very high protein content. Despite its high nutritional value and promotion efforts by regional researchers and breeders, tarwi is not a widely cultivated crop in its center of origin. In this review, we present the work carried out by public breeding programs of L. mutabilis at national agricultural research institutes, universities, and other institutions in Ecuador, Peru, and Bolivia. The main breeding method used in the Andes to improve local landraces has been mass selection to adapt lines to specific environments. At least 25 cultivars or ecotypes have been selected and released over the last 40 years using this breeding system. Nevertheless, breeders are currently struggling to develop new varieties that are high yielding, suitable for mechanized harvesting, have a low content of alkaloids or other anti-nutritional properties, and resistant to anthracnose (Colletotrichum acutatum). Therefore, it is necessary to reassess the potential of this crop and invest in its research to incorporate new techniques and breeding strategies to optimize the development of new varieties in the Andes which address the current cultivation challenges of the species.

ASSESSMENT OF PLANTS’ LEAF EXTRACTS FOR ANTIFUNGAL ACTIVITY AGAINST THE FUNGUS COLLETOTRICHUM ACUTATUM IN CHILI (CAPSICUM ANNUUM L.)

In horticultural crops, including chili, the wide use of synthetic fungicides has manifested to control anthracnose disease; however, these chemicals have some side effects to deal with. In reducing the dependency on synthetic fungicides, other alternatives and means need searching to prevent pathogenic fungi. Therefore, the presented study sought to examine plant extracts with the potential to inhibit the growth of pathogenic fungi Colletotrichum acutatum, the causal organism of anthracnose disease in chili. This study collected 20 potential plant species for the investigation. The leaf extraction used the maceration method in methanol and n-hexane. The contents of chemical compounds sustained the GC-MS analysis. All the leaf extracts tested for their bioactivity underwent the colony method and diffusion well. The results revealed that out of 20 types of plants, six plant species were capable of inhibiting the growth of C. acutatum fungi, including Piper nigrum, Piper ornatum, Piper retrofractum, Ficus septica, Samanea saman, and Tithonia diversifolia. The leaf extract of F. septica has the highest inhibition rate (81.11%) for the growth of C. acutatum compared with other plant leaf extracts. The GC-MS analysis of the F. septica leaf extract showed the presence of 15 types of metabolite constituents, with nine having antimicrobial activities.

Modelling the In Vitro Growth of Phytopathogenic Filamentous Fungi and Oomycetes: The Gompertz Parameters as Robust Indicators of Propolis Antifungal Action.

Propolis is a resinous mixture produced by honeybees, mainly from plant exudates. With a rich chemical composition including many phenolic compounds, mostly responsible for its biological properties, namely antimicrobial ones, propolis may be a promising alternative to synthetic pesticides. The study of propolis from the south of Portugal and of its potential against phytopathogenic agents are still very recent and different methodological approaches hinder a comparison of efficacies. In this context, we aimed to test the value of a mathematical model for the multiparametric characterization of propolis' antifungal action on solid medium assays. An ethanol extract (EE) of a propolis sample harvested in 2016 from Alves (A16) was characterized in terms of phenolic composition and antimicrobial potential against five phytopathogenic species. A16.EE (500-2000 µg/mL) inhibited the mycelial growth of all the species, with Phytophthora cinnamomi and Biscogniauxia mediterranea being the most susceptible and Colletotrichum acutatum being the least affected. The Gompertz mathematical model proved to be a suitable tool for quantitatively describing the growth profiles of fungi and oomycetes, and its parameters exhibit a high level of discrimination. Our results reveal that propolis extracts may have potential applications beyond traditional uses, particularly within the agri-food sector, allowing beekeepers to make their businesses more profitable and diversified.

Blueberry floral probiotics: nectar microbes inhibit the growth of Colletotrichum pathogens.

To identify whether microorganisms isolated from blueberry flowers can inhibit the growth of Colletotrichum, an opportunistic plant pathogen that infects flowers and threatens yields, and to assess the impacts of floral microbes and Colletotrichum pathogens on artificial nectar sugars and honey bee consumption. The growth inhibition of Colletotrichum (Colletotrichum acutatum, Colletotrichum fioriniae, and Colletotrichum gloeosporioides) was screened using both artificial nectar co-culture and dual culture plate assays. All candidate nectar microbes were screened for antagonism against a single C. acutatum isolate. Then, the top four candidate nectar microbes showing the strongest inhibition of C. acutatum (Neokomagataea thailandica, Neokomagataea tanensis, Metschnikowia rancensis, and Symmetrospora symmetrica) were evaluated for antagonism against three additional C. acutatum isolates, and single isolates of both C. fioriniae and C. gloeosporioides. In artificial nectar assays, single and three-species cultures inhibited the growth of two of four C. acutatum isolates by ca. 60%, but growth of other Colletotrichum species was not affected. In dual culture plate assays, inhibition was observed for all Colletotrichum species for at least three of four selected microbial antagonists (13%‒53%). Neither honey bee consumption of nectar nor nectar sugar concentrations were affected by any microbe or pathogen tested. Selected floral microbes inhibited growth of all Colletotrichum species in vitro, although the degree of inhibition was specific to the assay and pathogen examined. In all microbial treatments, nectar sugars were preserved, and honey bee preference was not affected.

In Vitro Studies of Endophytic Bacteria Isolated from Ginger (Zingiber officinale) as Potential Plant-Growth-Promoting and Biocontrol Agents against Botrytis cinerea and Colletotrichum acutatum

Agriculture currently confronts a multitude of challenges arising from the excessive utilization of chemical pesticides and the proliferation of phytopathogenic fungi strains that exhibit resistance to commonly employed active compounds in the field. Botrytis cinerea and Colletotrichum acutatum are phytopathogenic fungi that inflict substantial economic losses within agriculture and food due to their high impacts on crops both pre- and post-harvest. Furthermore, the emergence of fungal strains that are resistant to commercial fungicides has exacerbated this problem. To explore more environmentally sustainable alternatives for the control of these pathogens, an investigation into the endophytic bacteria associated with ginger (Zingiber officinale Rosc.) was conducted. The primary focus of this study involved evaluating their inhibitory efficacy against the fungi and assessing their potential for promoting plant growth. The endophytic bacteria genera Lelliottia, Lysinibacillus, Kocuria, Agrococcus, Acinetobacter, Agrobacterium, Zymobacter, and Mycolicibacterium were identified. All isolates showed remarkable in vitro antagonistic ability against B. cinerea (&gt;94%) and C. acutatum (&gt;74%). Notably, the Lelliottia amnigena J29 strain exhibited a notable proficiency in producing extracellular enzymes and indole compounds (IAA), solubilizing phosphate and potassium, and forming biofilm. Furthermore, the Lysinibacillus capsici J26, Agrococcus citreus J28, and Mycolicibacterium sp. J5 strains displayed the capacity for atmospheric nitrogen fixation and siderophore production. These findings underscore the agricultural and biotechnological potential of endophytic bacteria derived from ginger plants and suggest the feasibility of developing alternative approaches to manage these two phytopathogenic fungi.

First report of Colletotrichum fioriniae Causing Anthracnose Disease of Rhus chinensis in China.

Rhus chinensis, a tree of major economic importance in China, belongs to the Anacardiaceae. It is the summer host of the aphidMelaphis chinensis which produces a leaf gall utilized for medicinal purposes (Li et al. 2022). In August 2021 and June 2022, dark brown spots were observed on young branches of R. chinensis in Wufeng, Hubei province, China. The plantations of R. chinensis in Wufeng County had different degrees of disease. We focused our survey on three plantations, each with an area of 1.5 hectares and 1600 R. chinensis plants per hectare, and the incidence of the disease was found to be around 70%. Symptoms began as small brown spots that expanded with time and eventually led to large, irregular, dark brown and sunken lesions. Under high temperature and humidity, orange conidiomata appeared on top of the lesions. As the disease progressed, branches rotted, broke, and leaves died and dropped, eventually causing the death of trees. The fungus was isolated from infected branches. Branch pieces were cut and surface disinfested in 75% (v/v) alcohol for 30 sec, then sterilized in 4% sodium hypochlorite for 1 min, and washed three times with sterile distilled water before incubated on potato dextrose agar (PDA) at 25℃.Ten isolates were obtained by a single-spore culturing method, considering HTK-3 isolate showed more pathogenic and grew faster than other isolates, it was selected for further research. After culturing for 7 days on PDA medium, the colony of isolate HTK-3 was cottony with white-to-gray aerial mycelium. The mycelial growth rate was 8.7 mm/day at 25℃. Conidia were single-celled, colorless, smooth-walled, fusiform with acute ends, and measured 7.7 to 14.3 × 3.2 to 5.3μm (mean 11.8 ± 1.3 to 4.2 ± 0.5μm, n = 50). Appressoria were single, medium brown, ovate to ellipsoid, 5.8 to 8.5× 3.7 to 6.1μm (mean 7.2 ± 0.7 × 4.9 ± 0.4 μm, n=50). Microscopic examinations showed conidia of the HTK-3 were hyaline, aseptate, and sub-cylindrical, with obtuse apices and tapering bases. Mycelium of which was hyaline, branched and septate. Based on these morphological features, the fungus was tentatively identified as belonging to Colletotrichum acutatum species complex (Damm et al. 2012). For molecular identification, the ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), beta-tubulin 2 (TUB2), and actin (ACT) were amplified and sequenced (Liu et al., 2022). The obtained sequences were deposited into the GenBank [accession numbers: OP630818 (ITS); OP649736 (GAPDH); OP649735 (TUB2); OP649738 (CHS-1); OP649737 (ACT)]. For all of these genes, isolates HTK-3 had a 99-100% similarity to multiple C. fioriniae accessions. A maximum likelihood tree was generated from a multiple sequence alignment of reported isolates (Liu et al. 2022) and HTK-3 was identified as C. fioriniae. To fulfill Koch's postulates, ten healthy branches were inoculated with 5-mm-diameter mycelial plugs of each of ten fungal isolates (Wang et al., 2022). PDAs plug without mycelium was used as control. Six days post-inoculation, all branches developed anthracnose symptoms similar to those observed in the field, while the control remained healthy. The pathogenicity tests were repeated twice with the same results. C. fioriniae was re-isolated from the disease branches and the morphology was consistent with original, fulfilling Koch's postulates. The species of C. fioriniae has been reported to cause severe anthracnose of many species of plants (Eaton et al. 2021). To our knowledge, this is the first report of C. fioriniae as a pathogen of R. chinensis in China. The results will help to target the screening of control agents and provide guidance for disease prevention and control.

Polyoxygenated cyclohexenoids from the endophytic fungus, Aspergillus aculeatus BCC69431 and biological properties

Four undescribed polyoxygenated compounds, including aspergilosidic acid, aspergilosidols A, B, and aspergilosidone, along with seven known compounds were isolated from the endophytic fungus, Aspergillus aculeatus BCC69431. The chemical structures with their absolute stereochemistry were determined by NMR spectroscopic information, CD spectral data, and chemical reaction. The isolated compounds were evaluated for biological activities such as antimalarial, antimycobacterial, antibacterial, anti-plant pathogenic fungal activities, as well as cytotoxicity against cancerous (MCF-7 and NCI–H187) and non-cancerous (Vero) cells. Only violaceoid A (8) exhibited antimalarial against Plasmodium falciparum, K1 strain (IC50 9.62 μM), antibacterial against Staphylococcus aureus (MIC 12.50 μg/mL), and antifungal against Colletotrichum acutatum (MIC 50.0 μg/mL) activities without significant cytotoxicity. In addition, himeic acid G (11) showed anti-Mycobacterium tuberculosis activity with MIC value of 25.0 μg/mL with no cytotoxicity at the maximum tested concentration (50 μg/mL), while microsphaerone C (9) possessed cytotoxicity against NCI–H187 with IC50 value of 1.88 μM.

Genes and Loci Controlling the Resistance of Strawberry (F. ananassa Duch.) to Pathogens

Strawberry (F. ànanassa) is one of the most popular and widespread berry crops in the world. Strawberry diseases are the main limiting factor that seriously affects the yield of plants and leads to economic losses. Most strawberry cultivars are very sensitive to pathogenic fungi and bacteria, such as Colletotrichum acutatum (anthracnose root), Colletotrichum gloeosporioides (crown rot), Fusarium oxysporum fragariae (fusarium wilt), Phytophthora fragariae (red stele root rot), Verticillium dahlia (verticillium wilt), Botrytis cinerea (grey mould) and Xanthomonas fragariae (angular leaf spot). Currently, due to the rapid development of molecular genetics in general, and in particular knowledge related to the organization of genomes of agricultural crops, the use of DNA technologies in strawberry breeding has become possible. To identify disease resistance loci, the most popular approach was to map the loci of quantitative traits (QTL). Accurate evaluation of heritability and the number, location and effects of loci controlling traits are the basis for strategic decisions in breeding programs. The purpose of this review is to present up-to-date data on known genes and quantitative trait loci (QTL) that control the resistance to the most common strawberry pathogens.

The effectiveness of the mixture of basil and betel leaves methanol extracts to suppress Colletotrichum acutatum, the causal agent of anthracnose disease on postharvest cayenne chili

Abstract. Nasahi C, Ramadhanty S, Sudarjat S, Kurniadie D, Bari IN, Subakti-Putri SN. 2023. The effectiveness of the mixture of basil and betel leaves methanol extracts to suppress Colletotrichum acutatum, the causal agent of anthracnose disease on postharvest cayenne chili. Biodiversitas 24: 5513-5522. Anthracnose, caused by Colletotrichum acutatum, is a major disease in the cultivation of chili plantations in Indonesia. It is usually controlled by farmers using synthetic fungicides with many adverse effects. Therefore, botanical fungicides are applied as alternative control techniques. This research aimed to examine the effect of mixed methanol extract of basil (Ocimum basilicum) and betel leaves (Piper betle) against C. acutatum on cayenne chili (Capsicum annuum). The method used was a randomized block design (RBD) with thirteen treatments and three replications. The results showed that combining basil and betel leaf methanol extracts suppressed C. acutatum. Furthermore, the most effective treatment was a mixture of 1.5% of each of the extracts, which suppressed the development of the disease at 62.07%. The GC-MS profiling of the basil and betel leaf methanol fraction indicated the presence of bioactive compounds that have antifungal effects against anthracnose.

First Report of Colletotrichum fioriniae Infecting Hop (Humulus lupulus) in Michigan.

In July of 2020, a hop (Humulus lupulus L.) grower in Berrien County, Michigan submitted 'Chinook' leaf samples to MSU Plant & Pest Diagnostics. The leaves were covered in small, tan colored lesions, with a small chlorotic halo with an approximate diameter of 5 mm. The grower reported that foliar lesions were in the lower 2 m of the fully developed hop canopy. Disease incidence and severity were estimated at approximately 20% and 5 to 10%, respectively. After incubation at 100% relative humidity, acervuli with orange spore masses and a few setae were present. A pure culture was obtained from these sporulating lesions using water agar. The isolate was hyphal tipped onto potato dextrose agar (PDA) and stored in a glycerol-salt solution at -80o C (isolate CL001) (Miles et al. 2011). On PDA, cultures displayed gray growth on the top of the colony and a red color on the underside of the Petri dish. After 14 days, acervuli with no setae appeared exuding orange conidial masses on the surface of the culture. Conidia were hyaline, aseptate, smooth-walled and rounded at the ends and measured on average 15.89 μm (13.81 to 16.91 μm) × 7.26 (6.82 to 8.41 μm) (n = 20). The color and size of the conidia matched other descriptions of C. acutatum sensu lato (Damm et al. 2012). Four loci (ITS/515 bp - OQ026167, GAPDH/238 bp - OQ230832, CHS1/228 bp -OQ230830, and TUB2/491 bp - OQ230831) were amplified from isolate CL001 (using the primers ITS1/ITS4, GDF1/GDR1, CSH-79f/CHS-354R, and T1/Bt-2b, respectively) and had 100% pairwise identity with C. fioriniae 125396 (JQ948299, JQ948629, JQ948960, JQ949950, respectively, Damm et al. 2012). The GAPDH, CSH1, and TUB2 sequences from isolate CL001 were trimmed, concatenated and aligned with 31 different members of Colletotrichum acutatum sensu lato and C. gloesporioides 356878 (Damm et al. 2012; Kennedy et al. 2022). The alignment was then used to produce a maximum likehood phylogenetic tree using Geneious Prime (Biomatters Ltd.) with the PHYML add on using the HKY + G model (G = 0.34) (Guindon et al. 2010). Isolate CL001 had the closest similarity to C. fioriniae with a bootstrap value of 100. Pathogenicity tests were performed on 2 month-old 'Chinook' hop plants. Twelve plants were inoculated with 50 ml of a conidial suspension (7.95 x 106 conidia/ml) of isolate CL001 (n = 6) or water (n = 6) using a spray bottle until runoff. Inoculated plants were sealed in clear plastic bags and grown in a greenhouse at 21o C with a photoperiod of 14 h. After 7 days, lesions appeared on the hop plants inoculated with CL001, but no symptoms appeared on the water inoculated hop plants. Lesions with a chlorotic halo were observed but they were smaller than field lesions and no setae were present (approx. 1 mm in diam.). Leaves were surface sterilized (0.3% sodium hypochlorite solution for 15 s and then rinsed three times) and the leading margin of the lesions or healthy tissue (water control) were placed on 1% ampicillin amended PDA. Fungal isolates on PDA morphologically matched C. fioriniae were recovered from all CL001-inoculated plants. No C. fioriniae isolates were recovered from the water-inoculated plants. Based on conidial morphology, the four loci, and the phylogenetic tree, isolate CL001 was identified as C. fioriniae. This is the first report of Colletotrichum fioriniae (syn = Glomerella acutata var. fioriniae Marcelino & Gouli) infecting common hop and further investigation is needed to determine if management is needed for this pathogen.

Defence responses triggered during the plant-pathogen interaction between strawberry (Fragaria x ananassa) and Colletotrichum acutatum

Strawberry (Fragaria x ananassa Duch.) production is an important economic activity in Argentina. Anthracnose, caused by Colletotrichum spp., is the most destructive fungal disease for strawberry and causes a huge economic loss every year. The gene-regulatory networks and metabolic pathways involved in the interaction between Fragaria ananassa and Colletotrichum acutatum are poorly understood. Sixteen strawberry cultivars were characterized in a local strain, M11, of C. acutatum, and the results showed that the Camarosa and Pajaro cultivars were the most tolerant and susceptible, respectively. Metabolic, biochemical and molecular analyses were used to study pathogen and strawberry interaction. The results showed that (1) after being infected by the pathogen, the Camarosa cultivar showed early induction of stomatal closure, callose and lignin accumulation; (2) the metabolic profile of the Camarosa cv. infected by C. acutatum showed high levels of carbohydrate accumulation, while the Pajaro cv. was characterized by accumulation of amino acids and their derivatives; (3) the expression of defence-related genes was induced earlier in the Camarosa cv. The above results demonstrated that an early and complex network of defence responses is triggered in the tolerant cultivar (Camarosa cv.), when infected by C. acutatum. Altogether, these results led us to expand the boundaries of knowledge of the metabolic pathways and gene-regulatory networks involved in the interaction between strawberry and Colletotrichum acutatum.