First Report of Colletotrichum chrysophilum Causing Anthracnose on Nance (Byrsonima crassifolia) in Mexico.

Nance fruit [Byrsonima crassifolia (L.) HBK] is a native crop widely distributed in Mexico and South America (Medina-Torres et al. 2018). It has been reported that nance is a good source of active compounds with anti-inflammatory, neuropharmacological and antioxidant effects. In 2022, the annual production of nance fruit in Mexico was of 7,713.13 tons and average yield of 5.64 t/ha with economic value of 51,952.66 million pesos (SIAP, 2022). This production generated significant economic income for the communities at a local, regional, and national level. In January 2023, irregular necrotic spots were observed on leaves and fruit of nance in an orchard of 50 nance trees located in San Sebastián Nopalera (16°54'52.73"N; 97°47'50.35"W), Oaxaca, Mexico. The incidence of the disease ranged from 50 to 60% of the trees. Infected fruit first showed dark-brown lesions with defined borders that coalesced to form large necrotic area. Isolates were purified by single spore isolation method (Choi et al. 1999).Colletotrichum strains were grown in PDA medium and five monoconidial isolates were obtained. A representative sample was selected (CNC-NP3) and deposited in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. Colony on PDA was white with sparse aerial mycelium, and the center was dark grey with abundant acervuli. Conidia (n = 100) were hyaline, aseptate, cylindrical with rounded apex, 13.5 to 15.2 × 4.3 to 5.1 µm. Appressoria (n = 20) were terminal or lateral, obovoid to clavate and some with slightly lobed, 9.9 to 11.6 × 5.3 to 6.6 µm. Based on the morphology, the isolate was identified as belonging to the Colletotrichum gloeosporioides species complex (Jayawardena et al. 2016). The representative isolate CNC-NP3 was identified by multilocus phylogenetic analysis using nucleotide sequences of internal transcribed spacer (ITS), actin (ACT), ß-tubulin (TUB2), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (Jayawardena et al. 2016). The sequences were deposited in GenBank (accessions nos. OQ861102 (ITS), OQ870548 (ACT), OQ870549 (TUB2), OQ870550 (GAPDH). The phylogenetic analysis was carried out by Maximum likelihood method using concatenated sequences of ITS, ACT, TUB2 and GAPDH genes (Kozlov et al. 2019). The multilocus phylogenetic analysis revelated clearly the isolate CNC-NP3 as Colletotrichum chrysophilum. To confirm pathogenicity of CNC-NP3, 30 healthy fruits were inoculated. Fifteen disinfected nance with wounds and fifteen nance without wounds were inoculate with 10 µL of conidial suspension (1×105 spores/mL) from 7-day old culture. And controls were inoculated using sterile distilled water. Fruits were placed in a moist chamber covered with plastic bag at 25 °C for 48 h to maintain high humidity. After 4 days the inoculation sites development symptoms that were identical to those initially observed in the field, whereas the control group remained symptomless. The pathogenicity test was performed twice, with the same results. The pathogen was re-isolated from the lesion to fulfill Koch's postulates. Currently, Colletotrichum chrysophilum has been reported causing anthracnose disease in several crops: apple in New York (Khodadadi et al. 2020), papaya in Mexico (Pacheco- Esteva et al.2022), Blueberry (Soares et al. 2022) and banana in Brazil (Astolfi et al. 2022). To our knowledge, this is the first report of anthracnose in Byrsonima crassifolia caused by Colletotrichum chrysophilum in Oaxaca, Mexico.

First Report of Colletotrichum chrysophilum Causing Papaya Anthracnose in Mexico.

Anthracnose, caused by Colletotrichum spp., is the most important fungal disease of papaya (Carica papaya L.) worldwide. In March 2020, mature papaya fruit (cv. Maradol) showing typical symptoms of anthracnose were observed in an orchard located in Pinotepa Nacional, Oaxaca, Mexico. Disease incidence of 100 papaya plants surveyed in the orchard was estimated at about 45%. Initially, small and water-soaked lesions appeared on the fruit surface, which later enlarged to circular sunken lesions with translucent light brown margins. On advanced infections, salmon-pink masses of spores were observed on the lesions. Twenty Colletotrichum-like colonies were consistently isolated on potato dextrose agar (PDA) medium at 25°C in the dark for 6 days and 10 monoconidial isolates were obtained. An isolate was selected as representative for further characterization. The isolate was deposited as CPM-H4 in the Culture Collection of Phytopathogenic Fungi of Plant Pathology Laboratory of the CIIDIR-Oaxaca of the Instituto Politécnico Nacional. On PDA, the colonies were initially light grey then later became dark grey with orange conidial masses after incubation for 7 days. Conidia (n= 50) were hyaline, aseptate, cylindrical with rounded ends, and measured 10.2 to 13.6 × 4.1 to 5.3 µm. Appressoria (n= 20) were mostly simple, solitary and smooth-walled, dark brown, and clavate, measuring 6.8 to 14.8 × 5.5 to 7.7 µm. Based on morphology, the isolate was tentatively identified as belonging to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For molecular identification, total DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), and partial sequences of actin (ACT), ß-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and chitin synthase (CHS-1) genes were amplified (Weir et al. 2012), and sequenced. The sequences were deposited in GenBank (accessions nos. OM965612 (ITS), OM959540 (ACT), ON065005 (TUB2), ON065003 (CHS-1), ON065004 (GAPDH). A phylogenetic tree based on Bayesian inference and including published ITS, ACT, TUB2, GAPDH, and CHS-1 sequence dataset for Colletotrichum spp. was constructed. The multilocus phylogenetic analysis clearly distinguished the isolate CPM-H4 as Colletotrichum chrysophilum. Pathogenicity of the fungus was verified on 10 healthy papaya fruits (cv. Maradol) without wounds. A drop of a conidial suspension (1 × 105 spores/ml) was placed on three locations on each fruit. Ten control fruit were treated in the same way but with sterilized water. The fruits were kept in a moist plastic chamber at 25°C and 12 h light/dark for 8 days. The pathogenicity test was repeated twice. All inoculated papaya fruits developed sunken necrotic lesions 6 days after inoculation, whereas no symptoms were observed on the control fruits. The fungus was consistently re-isolated only from the diseased fruits and found to be morphologically identical to the isolate used for inoculation, fulfilling Koch´s postulates. Colletotrichum chrysophilum has been previously reported to cause anthracnose on mango (Fuentes-Aragón et al. 2020a), avocado (Fuentes-Aragón et al. 2020b), and banana (Fuentes-Aragón et al. 2021) in Mexico; however, to our knowledge, this is the first report of C. chrysophilum causing papaya anthracnose in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species associated with papaya in Mexico through subsequent phylogenetic studies as well as to monitor the possible movement and distribution of this pathogen into other Mexican regions.

First Report of Colletotrichum siamense Causing Anthracnose of Guava (Psidium guajava) in Mexico.

Guava (Psidium guajava L.) is a small tree belonging to the Myrtaceae family and it is distributed worldwide in the tropical and subtropical areas. During the summer of 2019, symptoms of fruit anthracnose were observed on approx. 90% of 250 guava trees located in backyards in Juan Jose Rios, Sinaloa, Mexico. Lesions on guava fruit were irregular, necrotic, and sunken. On advanced infections, acervuli containing salmon-pink masses of spores were observed on the lesions. Twenty fruits were collected from 10 trees (2 fruits per tree). Colletotrichum-like colonies were consistently isolated on PDA medium and 20 monoconidial isolates were obtained. Four isolates were selected as representatives for morphological characterization, multilocus phylogenetic analysis, and pathogenicity tests. The isolates were deposited in the Culture Collection of Phytopathogenic Fungi of the Faculty of Agriculture of El Fuerte Valley at the Sinaloa Autonomous University (Accession nos. FAVF205-FAVF208). Colonies on PDA medium were flat with an entire margin, with abundant felty and white aerial mycelium, with pink conidial masses. Conidia (n= 100) were cylindrical, hyaline, aseptate, with ends rounded, and measuring 14.8 to 18.1 × 4.4 to 5.3 µm. Based on morphological features, the isolates were tentatively allocated in the C. gloeosporioides species complex (Weir et al. 2012). For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), as well as partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), chitin synthase (CHS-1) and glutamine synthetase (GS) genes were amplified by PCR (Weir et al. 2012), and sequenced. A phylogenetic tree based on Bayesian inference and including published ITS, GAPDH, TUB2, ACT, CHS-1, and GS data for Colletotrichum species was constructed. The multilocus phylogenetic analysis clearly distinguished the four isolates FAVF205-FAVF208 as C. siamense separating it from all other species within the C. gloeosporioides species complex. The sequences were deposited in GenBank (accessions nos. ITS: MW598512-MW598515; GAPDH: MW595216-MW595219; TUB2: MW618012-MW618015; ACT: MW595208-MW595211; CHS-1: MW595212-MW595215; and GS: MW618008-MW618011). Pathogenicity of the four isolates was verified on 40 healthy guava fruits. Twenty fruits were wounded with a sterile toothpick (2 mm in depth) and a mycelial plug (6 mm of diameter) was placed on each wound. Ten fruits inoculated with a PDA plug without mycelial growth served as controls. The fruit was kept in a moist plastic chamber at 25°C for 7 days. Pathogenicity of each isolate was tested with both non-wound and wound inoculation methods. The experiments were repeated twice with similar results. All inoculated fruits developed sunken necrotic lesions 4 days after inoculation, whereas no symptoms were observed on the control fruits. The fungi were consistently re-isolated only from the diseased fruits, fulfilling Koch´s postulates. Colletotrichum siamense has been previously reported on guava fruit in India (Sharma et al. 2015). However, to our best knowledge, this is the first report of C. siamense causing fruit anthracnose on guava in Mexico. Therefore, it is necessary to explore the diversity of Colletotrichum species on guava in detail through subsequent phylogenetic studies as well as to monitor the distribution of this pathogen into other Mexican regions.

Preliminary Bioactivity Assessment of Myrothecium Species (Stachybotryaceae) Crude Extracts against Aedes aegypti (Diptera: Culicidae): A First Approach from This Phytopathogenic Fungi.

Mosquitoes, as insect vectors, play a crucial role in transmitting viruses and parasites, leading to millions of human deaths in tropical and subtropical regions worldwide. This study aimed to evaluate the effects of ethanolic extracts of three species within the genus Myrothecium (M. roridum, M. dimerum, and M. nivale) on Aedes aegypti mosquito larvae to assess the inhibitory effect on growth and development, as well as to determine mortality. We quantify the average lethal concentrations and provide a qualitative characterization of the chemical groups responsible for their potential. Phytochemical screening revealed the presence of alkaloids, flavonoids, and terpenoids in the ethanolic extracts of the three fungal species. Tannins were found only in the extracts of M. dimerum and M. roridum. We observed a clear dependence of the effects of the crude extracts on mosquito larvae on the concentrations used and the duration of exposure. The toxic effect was observed after 48 h at a concentration of 800 ppm for both M. dimerum and M. nivale, while M. roridum showed effectiveness after 72 h. All three species within the genus Myrothecium exhibited 100% biological activity after 72 h of exposure at 600 ppm. At lower concentrations, there was moderate growth and development inhibitory activity in the insect life cycle. The study highlights the effectiveness of crude Myrothecium extracts in combating mosquito larvae, with effects becoming apparent between 48 and 72 h of exposure. This initial approach underscores the potential of the fungus's secondary metabolites for further in-depth analysis of their individual effects or synergies between them.

Engineered Metal Oxide Nanoparticles as Fungicides for Plant Disease Control.

Metal oxide nanoparticles are considered to be good alternatives as fungicides for plant disease control. To date, numerous metal oxide nanoparticles have been produced and evaluated as promising antifungal agents. Consequently, a detailed and critical review on the use of mono-, bi-, and tri-metal oxide nanoparticles for controlling phytopathogenic fungi is presented. Among the studied metal oxide nanoparticles, mono-metal oxide nanoparticles-particularly ZnO nanoparticles, followed by CuO nanoparticles -are the most investigated for controlling phytopathogenic fungi. Limited studies have investigated the use of bi- and tri-metal oxide nanoparticles for controlling phytopathogenic fungi. Therefore, more studies on these nanoparticles are required. Most of the evaluations have been carried out under in vitro conditions. Thus, it is necessary to develop more detailed studies under in vivo conditions. Interestingly, biological synthesis of nanoparticles has been established as a good alternative to produce metal oxide nanoparticles for controlling phytopathogenic fungi. Although there have been great advances in the use of metal oxide nanoparticles as novel antifungal agents for sustainable agriculture, there are still areas that require further improvement.

Microwave-Assisted Hydrodistillation of Essential Oil from Plectranthus amboinicus: Evaluation of Its Antifungal Effect and Chemical Composition.

Fusarium wilt, a vascular syndrome in a wide range of plants, is caused by the pathogen Fusarium Oxysporum. The objective of this investigation was to evaluate the antifungal effect of four essential oils (EOs) (Plectranthus amboinicus, Syzygium aromaticum, Lippia alba, and Rosmarinus officinalis), which were obtained by using microwave-assisted hydrodistillation (MAH), against F. oxysporum. The yield obtained from P. amboinicus with the use of MAH was 0.2%, which was higher than that of a conventional extraction; its extraction time was also shorter. For concentrations of 100 and 300 µL/L, P. amboinicus caused an inhibition rate of 27.2 and 55.7%, respectively, while S. aromaticum caused an inhibition rate of 23.1 and 87.3%, respectively. It was observed that increasing the concentration also increased the % inhibition rate. The extracts of L. alba and R. officinalis caused an inhibition rate of 14.5 and 14.7% at 500 µL/L, respectively, at 10 days of incubation, while at this concentration, P. amboinicus and S. aromaticum achieved 100%. The major chemical compounds of P. amboinicus were carvacrol (41.20%), o-cymene (11.61%), caryophyllene (11.45%), α-bergamotene (7.71%), and caryophyllene oxide (4.62%), and these monoterpene hydrocarbons were responsible for the biological activity. The essential oil of P. amboinicus in appropriate concentrations is a potent antifungal agent that could be used for the control of F. oxysporum.

Stability, Energetic, and Reactivity Properties of NiPd Alloy Clusters Deposited on Graphene with Defects: A Density Functional Theory Study.

Graphene with defects is a vital support material since it improves the catalytic activity and stability of nanoparticles. Here, a density functional theory study was conducted to investigate the stability, energy, and reactivity properties of NinPdn (n = 1-3) clusters supported on graphene with different defects (i.e., graphene with monovacancy and pyridinic N-doped graphene with one, two, and three N atoms). On the interaction between the clusters and graphene with defects, the charge was transferred from the clusters to the modified graphene, and it was observed that the binding energy between them was substantially higher than that previously reported for Pd-based clusters supported on pristine graphene. The vertical ionization potential calculated for the clusters supported on modified graphene decreased compared with that calculated for free clusters. In contrast, vertical electron affinity values for the clusters supported on graphene with defects increased compared with those calculated for free clusters. In addition, the chemical hardness calculated for the clusters supported on modified graphene was decreased compared with free clusters, suggesting that the former may exhibit higher reactivity than the latter. Therefore, it could be inferred that graphene with defects is a good support material because it enhances the stability and reactivity of the Pd-based alloy clusters supported on PNG.

Effectiveness of neutral electrolyzed water and copper oxychloride on fungi spores isolated from tropical fruits.

The aim of this study was to assess the antifungal effectiveness of neutral electrolyzed water (NEW) to inhibit the spore germination of post-harvest fungi common in fruits, determine the required available chlorine concentration (ACC) of NEW and to compare it with copper oxychloride (CO) and sterile distilled water (SDW) in vitro. This study evaluated the biological effectiveness of NEW to inactivate pure cultures of 11 different fungi obtained from post-harvest tropical fruits with anthracnose, rottenness or necrosis symptoms. A conidial solution of 1 × 104 spores/mL per culture was prepared and treated with a low, medium and high ACC of NEW (pH 7.0 ± 0.05, 12, 33 and 53 mg/L of ACC and ORP of 850 mV), CO at 0.3 g/L, or sterile distilled water as a control, for 3-, 5- and 10-min contact time. Spore germination of Alternaria alternata, Botrytis cinerea, Cladosporium australiense, Colletotrichum gloeosporioides and C. siamense, Fusarium solani and F. oxysporum, and Lasiodiplodia theobromae was inhibited in 100% by NEW at 12, 33 and 53 ppm ACC; 3,5 and 10 min contact time. Aspergillus niger and A. tamarii required 53 mg/L ACC to inhibit 100% of spore germination. NEW at 33 and 12 mg/L inhibited around 50% and <25% of A. niger spore germination, respectively. NEW at 53 mg/L ACC was the most efficient treatment against Rhizopus stolonifer but only inhibited spore germination in ∼25%. CO inhibited spore germination by 100% of A. alternata, B. cinerea, C. australiense, C. gloeosporioides, C. siamense and L. theobromae. However, CO inhibited <25% of spore germination of F. solani, F. oxysporum, A. niger, A. tamarii and R. stolonifer. NEW and CO had a significant effect on every fungus compared to a SDW treatment. SDW was the least effective treatment, followed by CO. NEW at 12 mg/L and 33 mg/L ACC were equally effective in eliminating the fungi, and more effective than CO. NEW at a concentration of 53 mg/L ACC was the most effective treatment. Results obtained in this study show that NEW has effectively inhibited spore germination of these species, and this treatment could be used as a substitute an ecological novel alternative to CO to avoid spore growth in the above-mentioned fruits.

Metal Nanoparticles as Novel Antifungal Agents for Sustainable Agriculture: Current Advances and Future Directions.

The use of metal nanoparticles is considered a good alternative to control phytopathogenic fungi in agriculture. To date, numerous metal nanoparticles (e.g., Ag, Cu, Se, Ni, Mg, and Fe) have been synthesized and used as potential antifungal agents. Therefore, this proposal presents a critical and detailed review of the use of these nanoparticles to control phytopathogenic fungi. Ag nanoparticles have been the most investigated nanoparticles due to their good antifungal activities, followed by Cu nanoparticles. It was also found that other metal nanoparticles have been investigated as antifungal agents, such as Se, Ni, Mg, Pd, and Fe, showing prominent results. Different synthesis methods have been used to produce these nanoparticles with different shapes and sizes, which have shown outstanding antifungal activities. This review shows the success of the use of metal nanoparticles to control phytopathogenic fungi in agriculture.

Asian Tiger Mosquito in Yucatan Peninsula: First Record of Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Campeche, Mexico.

This report describes the presence of Aedes albopictus Skuse (Diptera: Culicidae) in Yucatan Peninsula and represents the first record of the Asian tiger invasive mosquito in Campeche State, southeastern Mexico. We collected specimens using 11,326 ovitraps put into houses of urban and rural areas, as part of the entomological surveillance by the local Ministry of Health from January 2019 to February 2020. We found Ae. albopictus in five of the 12 municipalities of Campeche (San Francisco de Campeche, Tenabo, Hecelchakán, Calkíni and Escárcega). We record 68 positive ovitraps and 226 Ae. albopictus larvae. This finding increases the number of mosquito species recorded in Campeche, Mexico, and possibly the potential for 22 arbovirus transmission.

Argemone mexicana (Papaverales: Papavaraceae) as an Alternative for Mosquito Control: First Report of Larvicidal Activity of Flower Extract.

Culex quinquefasciatus Say (Diptera: Culicidae), an arboviral and filarial vector, is one of the most widespread mosquitoes in the world. The indiscriminate use of synthetic chemical insecticides has led to the development of resistance in mosquito populations worldwide. The effect of continuous exposure to crude extracts of Argemone mexicana, the Mexican poppy, on the development and growth stages of second-instar larvae of the mosquito was studied, along with qualitative chemical analysis of the different plant parts. Inhibition, mortality, and larval and pupal duration phases were assessed. Second-instar mosquito larvae were exposed to crude ethanol extracts of flowers, stems, and seeds. Flower extract exhibited the strongest larvicidal activity with LC50 and LC90 values after 24 h of exposure of 18.61 and 39.86 ppm, respectively, and 9.47 and 21.76 ppm after 48 h. Extracts from stem and seeds were significantly less effective. The flower extract registered a Growth Inhibition Index of 0.01 at 25 ppm, with stems and seeds registering 0.05 and 0.08, respectively, at 100 ppm (control group 1.02). Qualitative chemical analysis by thin-layer chromatography showed characteristic spots indicating the presence of alkaloids and flavonoids and phytochemical screening showed the presence of alkaloids, anthraquinones, flavonoids, tannins, and terpenoids in the various crude extracts. This is the first report of the effectiveness of an ethanol flower extract of A. mexicana on Cx. quinquefasciatus; it can be considered a promising alternative control for this mosquito species.

Bactericidal Activity of Chrysomya rufifacies and Cochliomyia macellaria (Diptera: Calliphoridae) Larval Excretions-Secretions Against Staphylococcus aureus (Bacillales: Staphylococcaceae).

The inhibitory effect of Chrysomya rufifacies (Macquart) and Cochliomyia macellaria (Fabricius) larval excretions-secretions (ES) on Staphylococcus aureus was determined using a portable colorimetric method without the need for any dedicated spectral instrument. Polystyrene 96 well microplates were used and 100 µl of the bacterial inoculum (5 × 105 CFU/ml) plus 100 µl of the dipteran exosecretions at different concentrations were added to each well. Subsequently, 50 µl of a 1% solution of the triphenyl tetrazolium chloride stain was added to each well to determine the bacterial viability. The color development in each well was measured with the ImageJ software S. aureus was exposed to different concentrations of the ES of both species individually. At a concentration of 800 ppm ES of C. rufifacies or Co. macellaria, bacterial growth was inhibited 97.45 ± 1.70% and 82.21 ± 1.88%, respectively. As expected, exposure to a lower concentration (i.e., 50 ppm) was less inhibitory (C. rufifacies ES, 77.65 ± 4.25% and Co. macellaria ES, 43.54 ± 4.63%). This study demonstrates for the first time the bactericidal activity of C. rufifacies and Co. macellaria ES against S. aureus. This finding is promising as it could result in the identification and synthesis of proteins capable of suppressing pathogen development in wounds. Additionally, the proposed method can simplify the use of expensive laboratory instruments for antimicrobial activity determination.

Effectiveness of Neutral Electrolyzed Water on Incidence of Fungal Rot on Tomato Fruits ( Solanum lycopersicum L.).

We assessed the effect of neutral electrolyzed water (NEW) on the incidence of rot on tomato ( Solanum lycopersicum L.) fruits inoculated with Fusarium oxysporum , Galactomyces geotrichum , and Alternaria sp. at sites with lesions. The inoculated fruits were treated with NEW at 10, 30, and 60 mg liter-1 active chlorine, with copper oxychloride fungicide, and with sterile distilled water (control) for 3, 5, and 10 min. In the experiment with F. oxysporum , 50 to 80% of the control fruits and 50 to 60% of the fruits treated with the fungicide exhibited symptoms of rot at the inoculated sites. The lowest incidence recorded was 30% for fruits treated with NEW at 60 mg liter-1 active chlorine with an immersion time of 5 min. In the experiment with G. geotrichum , incidence of rot on control fruits was 70 to 90%, and for treatment with fungicide rot incidence was 50 to 90%. NEW at 60 mg liter-1 active chlorine significantly reduced incidence of symptomatic fruit: only 30% of the inoculated fruits washed for 5 min had damage from rot. In the experiment with Alternaria sp., 60 to 90% of the fruits in the control group and 60 to 70% of the fruits in the fungicide group were symptomatic. The lowest incidence was recorded for the treatment in which the fruits were submerged in NEW with 60 mg liter-1 active chlorine for 3 min. In this group, 40 to 50% of the fruits exhibited symptoms of rot. These results were obtained 8 days after inoculation. NEW, with 60 mg liter-1 active chlorine, significantly reduced incidence of rot symptoms on fruits inoculated with one of the experimental fungi relative to the control (P ≤ 0.05). NEW at 60 mg liter-1 is effective in the control of fungal rot in tomatoes.