Naringin inhibits cisplatin resistance of ovarian cancer cells by inhibiting autophagy mediated by the TGF-ß2/smad2 pathway.

Background: Resistance to cisplatin (DDP) in patients with ovarian cancer (OC) poses a great challenge to improving the quality of life of patients. Past reports have revealed that naringin can induce apoptosis of OC cells and delay the occurrence of drug resistance in OC cells. However, the molecular role by which naringin inhibits DDP resistance in OC has not been definitively proven by researchers. The objective of this study is to investigate the effect of naringin on DDP resistance in OC cells and the specific mechanism of naringin mediating autophagy. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were selected to evaluate the role of naringin or DDP on the proliferation and apoptosis of human OC cells (SKOV3/A2780). The protein levels of Sequestosome 1 (SQSTM1/p62, hereinafter referred to as p62), microtubule-associated protein 1 light chain 3 (LC3), transforming growth factor-ß2 (TGF-ß2) and SMAD family member 2 (smad2) were detected with Western blotting assay. Immunofluorescence assay was also used to evaluate the level of LC3 in different groups of cells. Besides, functional analyses were performed in vivo. Results: Naringin was shown to promote DDP sensitivity and apoptosis of human OC DDP-resistant cell line (SKOV3/A2780-DDP cells). Significantly increased p62 expression and reduced LC3 expression were found in naringin-treated cells. The autophagy agonist, rapamycin, reversed the effect of naringin on the resistance of SKOV3-DDP cells to DDP. Naringin inhibited levels of TGF-ß2/smad2 pathway-related proteins, and regulated autophagy in SKOV3-DDP cells. In vivo experiments demonstrated that injection of naringin inhibited DDP resistance and autophagy in mice xenograft model. Conclusions: In summary, naringin inhibits DDP resistance in OC cells by inhibiting autophagy mediated by the TGF-ß2/smad2 pathway.

Colletotrichum caladii sp. nov. causing anthracnose leaf spot of Caladium × hortulanum (Araceae) in Florida, USA.

Caladium (Caladium × hortulanum) is an ornamental plant popular for its variable and colorful foliage. In 2020, plants showing leaf spots and blight, typical of anthracnose, were found in a field trial at the University of Florida's Gulf Coast Research and Education Center (UF/GCREC) in Wimauma, FL, USA. Leaf samples consistently yielded a Colletotrichum-like species with curved conidia and abundant setae production in the acervuli. The internal transcribed spacer (ITS), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase gene (gapdh), actin gene (act), chitin synthase 1 gene (chs-1), beta-tubulin gene (tub2), and histone3 gene (his3) were amplified and sequenced. Blastn searches in the NCBI GenBank database revealed similarities to species of the Colletotrichum truncatum species complex. Phylogenetic analyses using multi-locus sequence data supports a distinct species within this complex, with the closest related species being C. curcumae. Based on morphological and phylogenetic analyses, a new species of Colletotrichum, named C. caladii, is reported. Pathogenicity assays and subsequent isolation confirmed that this species was the causal agent of the disease.

Greenhouse gas emission characteristics and influencing factors of agricultural waste composting process: A review.

China, being a major agricultural nation, employs aerobic composting as an efficient approach to handle agricultural solid waste. Nevertheless, the composting process is often accompanied by greenhouse gas emissions, which are known contributors to global warming. Therefore, it is urgent to control the formation and emission of greenhouse gases from composting. This study provides a comprehensive analysis of the mechanisms underlying the production of nitrous oxide, methane, and carbon dioxide during the composting process of agricultural wastes. Additionally, it proposes an overview of the variables that affect greenhouse gas emissions, including the types of agricultural wastes (straw, livestock manure), the specifications for compost (pile size, aeration). The key factors of greenhouse gas emissions during composting process like physicochemical parameters, additives, and specific composting techniques (reuse of mature compost products, ultra-high-temperature composting, and electric-field-assisted composting) are summarized. Finally, it suggests directions and perspectives for future research. This study establishes a theoretical foundation for achieving carbon neutrality and promoting environmentally-friendly composting practices.

Relative contribution of ammonia-oxidizing bacteria and denitrifying fungi to N2O production during rice straw composting with biochar and biogas residue amendments.

Nitrous oxide (N2O) production is associated with ammonia-oxidizing bacteria (amoA-AOB) and denitrifying fungi (nirK-fungi) during the incorporation of biochar and biogas residue composting. This research examined the relative contribution of alterations in the abundance, diversity and structure of amoA-AOB and nirK-fungi communities on N2O emission by real-time PCR and sequence processing. Results showed that N2O emissions showed an extreme relation with the abundance of amoA-AOB (rs = 0.584) while giving credit to nirK-fungi (rs = 0.500). Nitrosomonas and Nitrosospira emerged as the dominant genera driving ammoxidation process. Biogas residue changed the community structure of AOB by altering Nitrosomonadaceae proportion and physiological capacity. The denitrification process, primarily governed by nirK-fungi, served as a crucial pathway for N2O production, unveiling the pivotal mechanism of biochar to suppress N2O emissions. C/N and NH4+-N were identified as significant parameters influencing the distribution of nirK-fungi, especially Micromonospora, Halomonas and Mesorhizobium.

Identification of sequence mutations in Phytophthora cactorum genome associated with mefenoxam resistance and development of a molecular assay for the mutant detection in strawberry (F. × ananassa).

Phytophthora crown rot (PhCR) caused by Phytophthora cactorum is one of the most damaging diseases of strawberry worldwide. Mefenoxam is one of the major fungicides currently used to manage PhCR. However, the emergence and spread of resistant isolates have made controlling the pathogen in the field problematic. In the present study, using whole genome sequencing analysis, mutations associated with mefenoxam-resistant isolates were identified in six different genomic regions of P. cactorum. The 95.54% reads from a sensitive isolate pool and 95.65% from a resistant isolate pool were mapped to the reference genome of P. cactorum P414. Four point mutations were in coding regions while the other two were in noncoding regions. The genes harboring mutations were functionally unknown. All mutations present in resistant isolates were confirmed by sanger sequencing of PCR products. For the rapid diagnostic assay, SNP-based high-resolution melting (HRM) markers were developed to differentiate mefenoxam-resistant P. cactorum from sensitive isolates. The HRM markers R3-1F/R3-1R and R2-1F/R2-1R were suitable to differentiate both sensitive and resistant profiles using clean and crude DNA extraction. None of the mutations associated with mefenoxam resistance found in this study were in the RNA polymerase subunit genes, the hypothesized target of this compound in oomycetes. Our findings may contribute to a better understanding of the mechanisms of resistance of mefenoxam in oomycetes since serves as a foundation to validate the candidate genes as well as contribute to the monitoring of P. cactorum populations for the sustainable use of this product.

Response characteristics of antibiotic resistance genes and bacterial communities during agricultural waste composting: Focusing on biogas residue combined with biochar amendments.

This research investigated biogas residue and biochar addition on antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and changes in bacterial community during agricultural waste composting. Sequencing technique investigated bacterial community structure and ARGs, MGEs changes. Correlations among physicochemical factors, ARGs, MGEs, and bacterial community structure were determined using redundancy analysis. Results confirmed that biochar and biogas residue amendments effectively lowered the contents of ARGs and MGEs. The main ARGs detected was sul1. Proteobacteria and Firmicutes were the main host bacteria strongly associated with the dissemination of ARGs. The dynamic characteristics of the bacterial community were strongly correlated with pile temperature and pH (P < 0.05). Redundancy and network analysis revealed that nitrate, intI1, and Firmicutes mainly affected the in ARGs changes. Therefore, regulating these key variables would effectively suppress the ARGs spread and risk of compost use.

Pseudocercospora pancratii Causing Leaf Spots on Commercial Blackberry (Rubus sp.) in Florida.

Blackberry (Rubus L. subgenus Rubus Watson) is a deciduous berry crop that is the fourth most economically important berry crop, and its production is expanding in the southeastern United States. However, since most commercially available cultivars were bred under temperate conditions, they are not always well adapted and could be threatened by new pathogen populations inhabiting subtropical areas. In 2017, plants showing purple or brown leaf spots and angular-to-irregular lesions on both leaf surfaces, with clusters of black conidiophores at the center, were observed in a field trial at the University of Florida's Gulf Coast Research and Education Center (UF/GCREC) in Wimauma, FL. A fungus resembling Cercospora/Pseudocercospora was isolated from the lesions. The ribosomal DNA internal transcribed spacers, the translation elongation factor 1-alpha, and the actin genes were amplified and sequenced. Based on the phylogenetic analysis, the closest related species was Pseudocercospora pancratii. Pathogenicity assays and subsequent reisolation confirmed that this species is the causal agent of the disease. Among eight cultivars screened, no complete resistance was found. However, 'Osage' was the least susceptible, and 'Kiowa' was the most susceptible. This study is the first report of P. pancratii causing leaf spots on blackberry worldwide, and it may help shape future research into disease epidemiology and management for a crop that is rapidly expanding but has very limited disease information currently available for Florida growers.

Characteristics of carbon, nitrogen, phosphorus and sulfur cycling genes, microbial community metabolism and key influencing factors during composting process supplemented with biochar and biogas residue.

Carbon (C), nitrogen (N), phosphorus (P), and sulfur (S) cycling functional genes and bacterial and fungal communities during composting with biochar and biogas residue amendments were studied. Correlations between microbial community structure, functional genes and physicochemical properties were investigated by network analysis and redundancy analysis. It was shown that the gene of acsA abundance accounted for about 50% of the C-related genes. Biogas residue significantly decreased the abundance of denitrification gene nirK. Biogas residues can better promote the diversity of bacteria and fungi during composting. Biochar significantly increased the abundance of Humicola. Redundancy analysis indicated that pile temperature, pH, EC were the main physicochemical factors affecting the microbial community. WSC and NO3--N have significant correlation with C, N, P, S functional genes. The research provides a theoretical basis for clarifying the metabolic characteristics of microbial communities during composting and for the application of biochar and biogas residues in composting.

Evaluation of the synergistic effects of biochar and biogas residue on CO2 and CH4 emission, functional genes, and enzyme activity during straw composting.

This study examined the effects of biochar, biogas residue, and their combined amendments on CO2 and CH4 emission, enzyme activity, and related functional genes during rice straw composting. Results showed that the biogas residue increased CO2 and CH4 emissions by 13.07 % and 74.65 %, while biochar had more obvious inhibition. Biogas residue addition enhanced functional gene abundance more than biochar. Biogas residue raised the methanogens mcrA gene by 2.5 times. Biochar improved the Acetyl-CoA synthase and ß-glucosidase activities related to carbon fixation and decreased coenzyme activities related to methanogens. Biochar and biogas residue combined amendments enhanced the acsB gene abundance for CO2 assimilation process and decreased methyl-coenzyme M reductase α subunit activity. Pearson correlation analysis indicated that organic matter was the significant variable affecting CO2 and CH4 emissions (P < 0.01). These results indicated biochar played significant roles in carbon loss and greenhouse emissions caused by biogas residue incorporation during composting.

Genome sequence of Gossypium anomalum facilitates interspecific introgression breeding.

Crop wild relatives are an important reservoir of natural biodiversity. However, incorporating wild genetic diversity into breeding programs is often hampered by reproductive barriers and a lack of accurate genomic information. We assembled a high-quality, accurately centromere-anchored genome of Gossypium anomalum, a stress-tolerant wild cotton species. We provided a strategy to discover and transfer agronomically valuable genes from wild diploid species to tetraploid cotton cultivars. With a (Gossypium hirsutum × G. anomalum)2 hexaploid as a bridge parent, we developed a set of 74 diploid chromosome segment substitution lines (CSSLs) of the wild cotton species G. anomalum in the G. hirsutum background. This set of CSSLs included 70 homozygous substitutions and four heterozygous substitutions, and it collectively contained about 72.22% of the G. anomalum genome. Twenty-four quantitative trait loci associated with plant height, yield, and fiber qualities were detected on 15 substitution segments. Integrating the reference genome with agronomic trait evaluation of the CSSLs enabled location and cloning of two G. anomalum genes that encode peroxiredoxin and putative callose synthase 8, respectively, conferring drought tolerance and improving fiber strength. We have demonstrated the power of a high-quality wild-species reference genome for identifying agronomically valuable alleles to facilitate interspecific introgression breeding in crops.

Effects of biochar and biogas residue amendments on N2O emission, enzyme activities and functional genes related with nitrification and denitrification during rice straw composting.

This study was carried out to determine the response characteristics of N2O emission, enzyme activities, and functional gene abundances involved in nitrification/denitirification process with biochar and biogas residue amendments during rice straw composting. The results revealed that N2O release mainly occurred during the second fermentation phase. Biogas residue amendment promoted N2O emission, while biochar addition decreased its emission by 33.6%. The nirK gene was abundant through composting process. Biogas residues increased the abundance of denitrification genes, resulting in further release of N2O. Biochar enhanced nosZ gene abundance and accelerated the reduction of N2O. Nitrate reductase (NR), nitrite reductase (NiR), N2O reductase (N2OR), and ammonia monooxygenase (AMO) activities were greatly stimulated by biochar or biogas residue rather than their combined addition. Pearson regression analysis indicated that N2O emission negatively correlated with ammonium and positively correlated with nosZ, nirK, 18S rDNA, total nitrogen, and nitrate (P < 0.05).

Silicon fertilizers, humic acid and their impact on physicochemical properties, availability and distribution of heavy metals in soil and soil aggregates.

It has been confirmed that silicon (Si) fertilizer and humic acid (HA) could effectively decrease the heavy metals in soil. Nonetheless, the impact of these additives on soil aggregate characteristics was ignored. Therefore, the effects of Si fertilizer, HA, and their combinations on the physicochemical characteristics, availability of heavy metals (Cu, Cd, Pb, Zn), and fraction changes in soils and soil aggregates were investigated in this research. The results showed that Si fertilizer and HA significantly modified soil properties such as soil pH, electrical conductivity total organic carbon, water-soluble organic carbon, and nitrate­nitrogen. HA and Si-HA (SHA) supplementation significantly decreased the availability of Cu, Cd, Pd, and Zn. Besides, there was no significant difference in physicochemical properties between soil and soil aggregates. The availability of Cu, Cd, Pd, and Zn in soil aggregates could be significantly inhibited by the addition of HA and SHA, and the content in microaggregates was greater than that in macroaggregates. After the addition of the three additives, the main fractions of heavy metals in different particle sizes were changed and eventually transformed to the residue state. These results indicated that Si fertilizer, HA, and SHA were influential in physicochemical properties and metal availability in soil aggregates. Therefore, it is of great scientific significance to study the impact of heavy metal pollution on the ecological environment in different aggregates, which will provide reference data for future sustainable management of heavy-metal polluted soils.

Activities of functional enzymes involved in C, N, and P conversion and their stoichiometry during agricultural waste composting with biochar and biogas residue amendments.

This experiment was carried out to explore the effects of biochar, biogas residue and their combination amendment on enzyme activities and their stoichiometry during agricultural waste composting. A comprehensive analysis of the variation in, and stoichiometric correlations between, ß-glucosidase (BG), N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), and alkaline phosphatase (AKP) were determined. The results showed that biochar, biogas residue, and their combined addition significantly increased those enzyme activities. The potential C:P and N:P acquisition activities represented by ln(BG): ln(AKP) and ln(LAP + NAG): ln(AKP), were significantly decreased with biogas residue addition. BG, NAG and LAP were significantly negatively correlated with temperature, organic matter and water-soluble carbon. Redundancy analysis also showed that moisture and water-soluble carbon were significantly related to the variations of enzyme activities. Biochar and biogas residue changed the characteristics of the composting substrate, thus affecting the activity and stoichiometry of functional enzymes involved in C, N and P cycling.

Multilocus Phylogenetic Analyses of Colletotrichum gloeosporioides Species Complex Causing Crown Rot on Strawberry in Florida.

Colletotrichum gloeosporioides is the causal agent of Colletotrichum crown rot of strawberry in the southern United States. Recent multigene studies defined C. gloeosporioides as a complex species comprised of 37 species. In our study, we phylogenetically characterized C. gloeosporioides isolates from strawberry and other noncultivated plants around strawberry fields. One hundred fifteen strawberry isolates and 38 isolates from noncultivated hosts were sequenced for five genomic regions: internal transcribed spacer, actin, calmodulin, chitin synthase, and glyceraldehyde-3-phosphate dehydrogenase. Phylogenetic analysis using the maximum likelihood and Bayesian inference methods, based on partition-specific models, revealed that most of the isolates in Florida (86%) were closely related to C. siamense, whereas 14 isolates were closely related to C. theobromicola (syn. C. fragariae), four isolates were C. fructicola, and three isolates were C. clidemiae. However, only the first three species were pathogenic to strawberry. Morphological characteristics evaluated show that mycelial growth of all species is approximately 5 mm/day, but colony morphology varies by species and incubation conditions. In vitro mating of the isolates demonstrated that C. fructicola is homothallic whereas C. siamense and C. theobromicola isolates are heterothallic. The biological importance of these different Colletotrichum species is currently being investigated to determine whether different management strategies are needed in strawberry production fields.

Development of a Multiplex High-Throughput Diagnostic Assay for the Detection of Strawberry Crown Rot Diseases Using High-Resolution Melting Analysis.

Rapid and accurate disease diagnosis is a prerequisite for an effective disease management program in strawberry production. In Florida, Colletotrichum spp., Phytophthora spp., and Macrophomina phaseolina are the primary microorganisms causing strawberry crown rot. Even though the diseases can be caused by different pathogens, symptoms are indistinguishable and equally devastating. To inform strawberry growers in a timely fashion of diagnostic results for effective deployment of chemical control practices, we developed a multiplex high-resolution melting (HRM) assay to rapidly and accurately detect the abovementioned pathogens. The multiplex HRM assays using three predesigned primer pairs showed high specificity for individual species by generating specific melting peaks without cross-reaction between primers or with other common strawberry pathogens. The amplification limit of the assay was 1 pg of Colletotrichum and Phytophthora and 100 pg of M. phaseolina DNA per 10-µl reaction. However, the presence of different melting peaks was observed in mixed DNA samples and was concentration and target DNA dependent. A crude DNA extraction protocol was developed to allow high-throughput screening by minimizing the inhibitory effects. Moreover, we applied the HRM assay to 522 plant samples and found high correlations between conventional pathogen isolation and HRM and between singleplex and multiplex assays. Altogether, this multiplex HRM assay is specific, cost effective, and reliable for the timely detection of strawberry crown rot pathogens.

First Report of Diaporthe phaseolorum Causing Stem Canker of Hemp (Cannabis sativa).

Hemp is an annual herbaceous plant that is used for its fiber and oil in a variety of commercial and industrial products. In Florida, it is currently being explored as a new specialty crop. During a field trial from October to January 2019 in Wimauma, FL, a stem canker was observed on up to 60% of three-month-old plants of 'Eletta Campana', 'Carmagnola Selezionata', and 'Tygra'. Symptoms started on the main stems with light-to-dark brown lesions of different sizes and shapes. Over time, the lesions coalesced into large necrotic areas and bore pycnidia. Isolations were made from diseased stem tissues on General Isolation medium (Amiri et al. 2018) after surface disinfestation (Marin et al. 2020). The plates were placed in a growth chamber at 25°C under a 12/12 photoperiod. A fungus with white, floccose, aerial mycelium and pycnidia producing alpha and beta conidia was consistently isolated. Three single spore isolates were chosen for identification and pathogenicity tests. Pycnidia on PDA were globose to irregular and ranged from 170 to 250 µm long (210 ± 2.5, n = 50) and 140 to 220 µm wide (180 ± 2.7, n = 50). The alpha conidia were unicellular, hyaline, ellipsoidal to fusiform and ranged from 5.3 to 7.7 µm long (6.5 ± 1.6, n = 50) and 1.5 to 4.6 µm wide (2.8 ± 1.8, n = 50). The beta conidia were hyaline, elongated, filiform, straight or curved and ranged from 10.2 to 17.7 µm long (16.1 ± 2.2, n = 50) and 0.5 to 1.8 µm wide (0.8 ± 0.2, n = 50). Perithecia were not observed. Based on morphological features, the fungus was similar to anamorphs of Diaporthe spp. (Santos et al. 2011; Udayanga et al. 2015). DNA from the same three isolates was extracted using the FastDNA kit, and the ribosomal internal transcribed spacer (ITS), ß-tubulin (TUB), and calmodulin (CAL) regions were amplified following Udayanga et al. (2014), and Sanger sequenced by Genewiz. Sequences were deposited in GenBank (accession no. MT497039 to MT497047 for ITS, TUB, and CAL). BLASTn searches revealed isolates 20-58, 20-59, and 20-60 were 96.34% identical to the epitype isolate D. phaseolorum AR4203 for ITS (KJ590738.1, 527 bp out of 547 bp), 100% for TUB (KJ610893.1, 459 bp out of 459 bp), and 100% for CAL (KJ612135.1, 522 bp out of 522 bp) (Udayanga et al. 2015). Their identity was confirmed by phylogenetic analyses using maximum likelihood and Bayesian inference methods. To complete Koch's postulates, pycnidia of the same three isolates were harvested and crushed in 2 mL Eppendorf tubes containing 0.01% Tween 20. Conidia suspensions were adjusted to 106 spores/mL. Three 5-week-old potted plants of 'Eletta Campana' and 'Carmagnola Selezionata' per isolate were inoculated using a 1 mL syringe with a needle by injecting 200 µL of the suspension into the stem. Plants were placed inside clear plastic bags for 48 h and maintained in the greenhouse. Control plants were injected with sterile deionized water and kept under the same conditions. The pathogenicity test was repeated once. Four weeks after inoculation, inoculated plants developed stem cankers from which the same pathogen was isolated, whereas controls remained healthy. To our knowledge, this is the first report of D. phaseolorum causing stem canker on hemp. This pathogen has been reported causing canker on sunflower and Phaseolus spp. (Gomzhina and Gannibal 2018; Udayanga et al. 2015; Vrandecic et al. 2009). This discovery may help shape future research into disease epidemiology and management for a crop in which very limited disease information is available at the moment.

Outbreak of Leaf Spot and Fruit Rot in Florida Strawberry Caused by Neopestalotiopsis spp.

Pestalotiopsis-like species have been reported affecting strawberry worldwide. Recently, severe and unprecedented outbreaks have been reported in Florida commercial fields where leaf, fruit, petiole, crown, and root symptoms were observed, and yield was severely affected. The taxonomic status of the fungus is confusing because it has gone through multiple reclassifications over the years. Morphological characteristics, phylogenetic analyses, and pathogenicity tests were evaluated for strawberry isolates recovered from diseased plants in Florida. Phylogenetic analyses derived from the combined internal transcribed spacer, ß-tub, and tef1 regions demonstrated that although there was low genetic diversity among the strawberry isolates, there was a clear separation of the isolates in two groups. The first group included isolates recovered over a period of several years, which was identified as Neopestalotiopsis rosae. Most isolates recovered during the recent outbreaks were genetically different and may belong to a new species. On potato dextrose agar, both groups produced white, circular, and cottony colonies. From the bottom, colonies were white to pale yellow for Neopestalotiopsis sp. and pale luteous to orange for N. rosae. Spores for both groups were five-celled with three median versicolored cells. Mycelial growth and spore production were higher for the new Neopestalotiopsis sp. isolates. Isolates from both groups were pathogenic to strawberry roots and crowns. However, the new Neopestalotiopsis sp. proved more aggressive in fruit and leaf inoculation tests, confirming observations from the recent outbreaks in commercial strawberry fields in Florida.

Mechanisms Underlying the Antidepressant Effect of Acupuncture via the CaMK Signaling Pathway.

The CaMK pathway has been proven to play an important role in regulating cognitive function and emotional response. Acupuncture through the CaMK pathway improves depression-like behavior and the molecular mechanism related to its antidepressant remains to be explored. In this study, we aimed to determine whether the ability of acupuncture at Baihui (GV20) and Shenting (GV24) points to treat depression is related to the regulation of key proteins in the CaMK pathway. A rat model of depression was induced by chronic unpredicted mild stress (CUMS). Model rats in the electroacupuncture group were subjected to acupuncture at the Baihui (GV20) and Shenting (GV24) acupoints once a day for 20 min. Model rats in the fluoxetine group were gavaged with fluoxetine (1.8 mg/kg). Immunohistochemistry and Western blotting assays were used to evaluate immunoreactivity for and the protein expression levels of CaMKII, CaMKIV, and CaM. The results showed that electroacupuncture had a significant effect in rats with depression. Electroacupuncture and fluoxetine regulated the expression of key proteins in the CaMK signaling pathway, which is related to depression, in the hippocampi of rats. This indicates that acupuncture at Baihui (GV20) and Shenting (GV24) may alleviate depressive symptoms and reduce work- and life-related burdens and stress by regulating the CaMK signaling pathway.

First report of sour rot of strawberry caused by Geotrichum candidum in the United States.

Strawberry (Fragaria x ananassa) is an important crop in the U.S., and Florida is the second major producer in the country. In January 2020, an unknown fruit rot was observed in two strawberry fields in Dover (seedling selection) and Plant City (cultivar Florida127), Florida. Disease incidence varied from less than 1% in one field to up to 15% in the second field during some harvests where over-ripe fruit were present. Affected fruit had a water-soaked soft rot with a sour smell, and sometimes with white mycelium on the fruit surface. Direct isolation was performed from symptomatic fruit from each area by touching the surface of a lesion with a sterile needle and streaking the fungus over general isolation medium (Amiri et al. 2018). The fungus was incubated at 25°C and 12-h photoperiod for five days. Four single-spore isolates (20-46 and 20-47 from Plant City; 20-49 and 20-50 from Dover) obtained from different colonies were grown on potato dextrose agar (PDA). Colonies were white to cream, flat, with a powdery surface, and had a characteristic sour odor. Hyphae were hyaline, septate, growing in a dichotomous pattern and often disarticulating into arthroconidia, which were unicellular, spherical to cylindrical, measuring 4.8 to 9.5 × 3.5 to 5.6 µm (n = 50). Based on the morphological characteristics, the fungus was identified as Geotrichum candidum (De Hoog et al. 1986). To confirm the identity of the isolates, the ribosomal internal transcribed spacer (ITS-rDNA) and the translation elongation factor 1-alpha (TEF1-α) gene regions were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), and EF1-728F/EF1-1567R (Carbone and Kohn 1999), respectively. Consensus sequences were deposited in the GenBank (accession numbers MT353975 to MT353978 for ITS-rDNA, and MT346367 to MT346370 for TEF1-α). The BLASTn analysis revealed 99% identity with reference sequences of G. candidum for ITS-rDNA (KU373122) and TEF1-α (MK397513). Phylogenetic analysis, including ITS sequences of G. candidum and other spp., obtained from GenBank, was performed using maximum likelihood and Bayesian inference methods, implemented in MEGA-X and MrBayes, respectively. The isolates were grouped within the G. candidum clade. The pathogenicity of the fungus (isolate 20-46) was evaluated in ripe and green strawberry fruit (cultivar Florida127). Non-wounded and superficially wounded (with a sterilized needle) fruit were inoculated with 20 µl of a spore suspension (1 × 107 spore/ml), prepared by washing the surface of a 5-day-old colony on PDA, with sterile deionized water (SDW) plus 0.01% (v/v) Tween 20. Wounded and non-wounded control fruit were treated with SDW. Fruit were maintained in moist containers at 25°C. Each treatment consisted of 12 fruit (three replicates, each containing four fruit) and was evaluated daily for 10 days. Symptoms of sour rot were visible as soon as 48 hr after inoculation, but only on ripe (100% incidence) and green (58% incidence) wounded fruit. Symptoms progressed to an intense water-soaking with the presence of a typical white mycelium on the surface. Control fruit remained symptomless. The fungus was successfully recovered from symptomatic fruit, fulfilling Koch's postulates. Although strawberry fruit rots caused by G. candidum have already been reported in Pakistan and China (Hussain et al. 2016; Ma et al. 2018), this is the first report of this species causing sour rot on strawberry in the U.S. The disease may be a minor problem on damaged or over-ripe fruit, but further studies might be needed to determine its importance, distribution, and potential strategies for control.

Anthracnose Fruit and Root Necrosis of Strawberry Are Caused by a Dominant Species Within the Colletotrichum acutatum Species Complex in the United States.

Strawberry anthracnose fruit rot and root necrosis, caused by Colletotrichum acutatum, are primary limiting factors in fruit production fields in the United States. Recent research focusing on the phenotypic and genetic characteristics of this species has shed light on the diversity of the C. acutatum species complex. In this study, we performed multilocus sequence analysis of four genetic loci to characterize 217 C. acutatum isolates collected over a 23-year period from symptomatic plant tissues of strawberry from six different states. The results revealed two Colletotrichum spp. (C. nymphaeae and C. fioriniae), with 97.7% of the isolate collection (212 of 217) belonging to C. nymphaeae as a dominant clonal linage, regardless of the isolation source. No correlation between species groups and geographical origins of the isolates was observed. Further sequence comparison between historical and contemporary isolates showed the same populations being widely distributed throughout the strawberry nurseries and production fields in the United States and Canada. Subsequently, a subset of 12 isolates representing different quinone-outside inhibitor fungicide resistance profiles from root or fruit tissue of strawberry was selected for comparison of pathogenicity on strawberry. In this test, isolates of different resistance groups or different isolation sources exhibited a similar degree of aggressiveness and caused indistinguishable symptoms on strawberry crowns (P = 0.9555 and 0.7873, respectively) and fruit (P = 0.1638 and 0.1141, respectively), although a significant difference among individual isolates was observed in detached-fruit assays (P = 0.0123). Separate pathogenicity tests using isolates of the two species revealed C. nymphaeae being more aggressive than C. fioriniae in infecting strawberry roots and crowns (P = 0.0073). Therefore, given the occurrence and pathogenicity of C. nymphaeae, this species is likely the sole cause responsible for strawberry anthracnose in the United States.