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.

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.

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.

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.

The Effects of Nutrition and Environmental Factors on Conidial Germination and Appressorium Formation of Phyllosticta citricarpa, the Causal Agent of Citrus Black Spot.

Citrus black spot, caused by Phyllosticta citricarpa, has been identified in Florida since 2010 and can reduce fruit yield and marketability. The conditions required for conidial germination have been poorly understood for P. citricarpa, limiting further biological studies. In this study, the effects of citrus juices, concentration, pH, various carbon and nitrogen sources, and environmental conditions were evaluated in vitro. All tested juices, especially 'Valencia' (>85%, P < 0.05), favored germination and appressorium formation, whereas sterile water rarely stimulated germination (<1%). The 'Valencia' juice effect was concentration and pH dependent, and the maximum rate was reached in 1.5% juice with pH of 3.4. Most carbon, nitrogen, or complex sources did not favor germination or appressorium formation, with the exception of potato dextrose broth. An incubation period of 18 to 24 h at 24°C was required for peak germination and appressorium formation. The further analysis of critical juice components using synthetic citrus juice revealed that sugars, salts, citric acid, and thiamine were most important for germination and appressorium formation (>80%, P > 0.05). These results provide a better understanding of fungal biology of P. citricarpa and a robust and convenient system for further applications such as screening for efficacious fungicides.

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.

Fitness, Competitive Ability, and Mutation Stability of Isolates of Colletotrichum acutatum from Strawberry Resistant to QoI Fungicides.

Quinone-outside inhibitor (QoI) fungicides are used to manage anthracnose of strawberry, caused by Colletotrichum acutatum. However, selection for resistance to QoI fungicides was first reported in 2013 in Florida and, subsequently, in strawberry nurseries and production areas across the United States and Canada. C. acutatum resistance to QoIs is associated with the G143A point mutation in the cytochrome b gene. This mutation is known to be associated with field resistance even at high rates of QoI. In this study, we investigated the relative fitness and competitive ability of QoI-resistant and -sensitive C. acutatum isolates. A fitness comparison did not indicate any difference between resistant and sensitive isolates in aggressiveness, spore production, and mycelial growth at different temperatures. Additionally, in the absence of selection pressure, resistant and sensitive isolates were equally competitive. Cultivation of QoI-resistant and QoI-sensitive isolates for four culture cycles in vitro in the absence of azoxystrobin showed that QoI resistance was stable. The observed lack of fitness penalties and stability of the G143A mutation in QoI-resistant C. acutatum populations suggest that the interruption and further reintroduction of QoI fungicides might not be an option for strawberry nurseries and fruit production areas. Further investigation of alternative chemical and nonchemical C. acutatum control practices, in addition to the integration of multisite fungicides, is needed to reduce the occurrence and distribution of QoI-resistant populations in strawberry fields.

Mating Type and Simple Sequence Repeat Markers Indicate a Clonal Population of Phyllosticta citricarpa in Florida.

Phyllosticta citricarpa, the citrus black spot pathogen, was first identified in Florida in March 2010. Subsequently, this pathogen has become established in Florida but can be easily confused with the endemic nonpathogenic citrus endophyte P. capitalensis. In this study, the mating-type (MAT) loci of P. citricarpa and P. capitalensis were identified via draft genome sequencing and were characterized at the structural and sequence levels. P. citricarpa was determined to have an idiomorphic, heterothallic MAT locus structure, whereas P. capitalensis was found to have a single MAT locus consistent with a homothallic mating system. A survey of P. citricarpa isolates from Florida revealed that only the MAT1-2 idiomorph existed in the Floridian population. In contrast, isolates collected from Australia exhibited a 1:1 ratio of MAT1-1 and MAT1-2 isolates. Development and analysis of simple sequence repeat markers revealed a single multilocus genotype (MLG) in the Floridian population (n = 70) and 11 MLG within the Australian population (n = 24). These results indicate that isolates of P. citricarpa from Florida are likely descendent from a single clonal lineage and are reproducing asexually. The disease management focus in Florida will need to be concentrated on the production and dispersal of pycnidiospores.

Baseline Sensitivity of Guignardia citricarpa Isolates from Florida to Azoxystrobin and Pyraclostrobin.

Citrus black spot (CBS), caused by Guignardia citricarpa, is an emerging disease in Florida. Fungicide applications are the main control measure worldwide. The in vitro activity and baseline sensitivity of G. citricarpa isolates to quinone outside inhibitor (QoI) fungicides (azoxystrobin and pyraclostrobin) were evaluated. The effective concentration needed to reduce mycelial growth or spore germination by 50% (EC50) was determined for 86 isolates obtained from Florida counties where CBS is found. The effect of salicylhydroxamic acid (SHAM) plus azoxystrobin and pyraclostrobin was also assessed for mycelial growth and conidial germination. The mean EC50 for mycelial growth for azoxystrobin was 0.027 µg/ml and that for pyraclostrobin was significantly lower at 0.007 µg/ml (P < 0.0001). Similarly, the mean EC50 for conidial germination for azoxystrobin was 0.016 µg/ml and that for pyraclostrobin was significantly lower at 0.008 µg/ml (P < 0.0001). There was no effect of SHAM on inhibition of mycelial growth or conidial germination by the QoI fungicides but SHAM slightly affected mycelium inhibition by pyraclostrobin. Cytochrome b was partially sequenced and three group 1 introns were found. One intron was immediately post G143, likely inhibiting resistance-conferring mutations at that site. It is surmised that the QoI resistance risk is low in the Florida G. citricarpa population.

qPCR Quantification of Pathogenic Guignardia citricarpa and Nonpathogenic G. mangiferae in Citrus.

Citrus black spot, a major citrus disease caused by Guignardia citricarpa, was recently introduced in Florida. The nonpathogenic fungal endophyte G. mangiferae is commonly found in the same citrus tissues as G. citricarpa. Quantitative polymerase chain reaction (qPCR) assays based on internal transcribed spacer (ITS)-1 genes were developed to detect, quantify, and distinguish between these morphologically similar organisms in environmental samples. The primer/probe sets GCITS and GMITS were more than 95% efficient in single-set reactions in complex environmental DNA samples. Detection of 10 fg of G. citricarpa and G. mangiferae DNA was possible. Pycnidiospore disruption resulted in detection of single pycnidiospores with 78 (59 to 102; 95% confidence interval [CI]) and 112 (92 to 136; 95% CI) ITS copies for G. citricarpa and G. mangiferae, respectively. Detection was from partially decomposed leaves where fruiting bodies cannot be morphologically distinguished. Temperature and wetting period have significant effects on Guignardia spp. pseudothecia production in leaf litter. Based on relative biomass or the proportion of nuclei detected, G. citricarpa and G. mangiferae respond more strongly to wetting period than temperature. This qPCR assay will provide additional epidemiological data on black spot in tissues where G. citricarpa and G. mangiferae are not easily distinguished.

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.

A G alpha subunit gene is essential for conidiation and potassium efflux but dispensable for pathogenicity of Alternaria alternata on citrus.

Heterotrimeric G proteins play a profound role in the recognition and transduction of extracellular signals in eukaryotic cells. We characterized the AaG alpha1 gene, encoding a fungal Class I G alpha subunit of the GTP-binding protein, in Alternaria alternata of citrus. Interruption of AaG alpha1 with a marker gene resulted in fungal transformants producing fewer conidia, becoming hypersensitive to KCl, and displaying elevated lipolytic and pectolytic activities. Expression of a functional copy of AaG alpha1 in a null mutant restored all altered phenotypes to the wild type. The AaG alpha1 mutants, whose conidia germinate normally, caused necrotic lesions on citrus indistinguishable from wild type. Application of cAMP, its inhibitors (atropine and theophylline), or 3-isobutyl-1-methylxanthine (IBMX) decreased conidiation in the fungal strains carrying a functional AaG alpha1. In contrast, conidial formation in the null mutants was restored by dibutyryl-cAMP or by a low concentration of cAMP or theophylline (1 mM). Unlike the oxidative stress-responsive AaAP1 transcription activator or the AaFUS3 mitogen-activated protein kinase (MAPK), AaG alpha1 is not required for cellular resistance to oxidative, osmotic, or chemical stress. AaFUS3 has also been demonstrated to be essential for conidial formation, suggesting a possible interaction between AaG alpha1 and AaFUS3 during conidiation even though expression of AaG alpha1 was not affected by AaFUS3 and vice versa. Inactivation of AaG alpha1 suppressed accumulation of transcripts of the AaAP1 gene and the AaHSK1 gene encoding a histidine kinase. These are novel features that have not been previously characterized to be associated with the GTP-binding protein.

The FUS3 MAPK signaling pathway of the citrus pathogen Alternaria alternata functions independently or cooperatively with the fungal redox-responsive AP1 regulator for diverse developmental, physiological and pathogenic processes.

Alternaria alternata, the fungus that causes citrus brown spot, invades its hosts primarily through the production and action of a host-selective ACT toxin that kills citrus cells prior to invasion. In this study, we show that, in the tangerine pathotype of A. alternata, a mitogen-activated protein kinase (MAPK)-mediated signaling pathway governs a number of biological functions, either separately or in a cooperative manner, with the AaAP1 gene encoding a transcription regulator. The reported MAPK is encoded by the AaFUS3 gene, which we show to be necessary for conidial development, resistance to copper fungicides, melanin biosynthesis, and particularly, for elaboration of the penetration process. In contrast, AaFUS3 negatively controls salt tolerance and production of several hydrolytic enzymes. AaFUS3 has no apparent role in the biosynthesis of host-selective toxin or in resistance to oxidative stress. Both AaAP1 and AaFUS3 are required for fungal resistance to 2,3,5-triiodobenzoic acid (TIBA), 2-chloro-5-hydroxypyridine (CHP), diethyl maleate (DEM), and many pyridine-containing compounds. A strain with mutations in both AaAP1 and AaFUS3 displayed an increased sensitivity to these compounds. Expression of the AaAP1 and AaFUS3 genes and phosphorylation of AaFUS3 were also induced by CHP, DEM, or TIBA. Expression of two genes coding for a putative MFS transporter was coordinately regulated by AaAP1 and AaFUS3. The AaAP1::sGFP (synthetic green fluorescent protein) fusion protein became localized in the nucleus in response to CHP or TIBA. Inactivation of the AaAP1 gene, however, promoted phosphorylation of AaFUS3. Taken together, our results indicate that A. alternata utilizes specialized or synergistic regulatory interactions between the AP1 and MAPK signaling pathways for diverse physiological functions.

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.