Jaimin S. Patel

The Arf GAP AGAP2 interacts with β-arrestin2 and regulates β2-adrenergic receptor recycling and ERK activation.

AGAP2 [Arf (ADP-ribosylation factor) GAP (GTPase-activating protein) with GTP-binding-protein-like, ankyrin repeat and PH (pleckstrin homology) domains] is a multidomain Arf GAP that was shown to promote the fast recycling of transferrin receptors. In the present study we tested the hypothesis that AGAP2 regulates the trafficking of β2-adrenergic receptors. We found that AGAP2 formed a complex with β-arrestin1 and β-arrestin2, proteins that are known to regulate β2-adrenergic receptor signalling and trafficking. AGAP2 co-localized with β-arrestin2 on the plasma membrane, and knockdown of AGAP2 expression reduced plasma membrane association of β-arrestin2 upon β2-adrenergic receptor activation. AGAP2 also co-localized with internalized β2-adrenergic receptors on endosomes, and overexpression of AGAP2 slowed accumulation of β2-adrenergic receptor in the perinuclear recycling endosomes. In contrast, knockdown of AGAP2 expression prevented the recycling of the β2-adrenergic receptor back to the plasma membrane. In addition, AGAP2 formed a complex with endogenous ERK (extracellular-signal-regulated kinase) and overexpression of AGAP2 potentiated ERK phosphorylation induced by β2-adrenergic receptors. Taken together, these results support the hypothesis that AGAP2 plays a role in the signalling and recycling of β2-adrenergic receptors.

Ex Vivo Application of Secreted Metabolites Produced by Soil-Inhabiting Bacillus spp. Efficiently Controls Foliar Diseases Caused by Alternaria spp.

ABSTRACT Bacterial biological control agents (BCAs) are largely used as live products to control plant pathogens. However, due to variable environmental and ecological factors, live BCAs usually fail to produce desirable results against foliar pathogens. In this study, we investigated the potential of cell-free culture filtrates of 12 different bacterial BCAs isolated from flower beds for controlling foliar diseases caused by Alternaria spp. In vitro studies showed that culture filtrates from two isolates belonging to Bacillus subtilis and Bacillus amyloliquefaciens displayed strong efficacy and potencies against Alternaria spp. The antimicrobial activity of the culture filtrate of these two biological control agents was effective over a wider range of pH (3.0 to 9.0) and was not affected by autoclaving or proteolysis. Comparative liquid chromatography-mass spectrometry (LC-MS) analyses showed that a complex mixture of cyclic lipopeptides, primarily of the fengycin A and fengycin B families, was significantly higher in these two BCAs than inactive Bacillus spp. Interaction studies with mixtures of culture filtrates of these two species revealed additive activity, suggesting that they produce similar products, which was confirmed by LC-tandem MS analyses. In in planta pre- and postinoculation trials, foliar application of culture filtrates of B. subtilis reduced lesion sizes and lesion frequencies caused by Alternaria alternata by 68 to 81%. Taken together, our studies suggest that instead of live bacteria, culture filtrates of B. subtilis and B. amyloliquefaciens can be applied either individually or in combination for controlling foliar diseases caused by Alternaria species.

Implementation of loop-mediated isothermal amplification methods in lateral flow devices for the detection of Rhizoctonia solani

Abstract Rhizoctonia solani causes destructive diseases in many crops throughout the world, resulting in significant yield and quality losses. Early detection of R. solani would facilitate deployment of timely disease management strategies and prevention of spread of asymptomatic infected plant material. The aim of this study was to develop a simple, rapid and sensitive loop-mediated isothermal amplification (LAMP) method for the detection of R. solani in plants and soil samples. LAMP primers, based on the internal transcribed spacer DNA sequence, were designed for detecting most anastomosis groups of R. solani. Using these primers, a LAMP protocol was developed, which in sensitivity tests was shown to detect very low levels of DNA of R. solani and R. zeae, but not R. oryzae. This LAMP protocol successfully detected R. solani on inoculated plant tissues and soil samples. For onsite application in the field, the LAMP protocol was implemented in a generic anti-biotin and anti-fluorescein antibody-based LFD. LAMP reactions were performed using biotin-labelled primers, which were hybridized with a fluorescein amidite (FAM)-labelled hybridization probe and detected with the LFD. This LAMP-LFD successfully detected R. solani in infected plant tissues. The LAMP-LFD procedure presented here is simple and rapid, and is comparable to quantitative real-time PCR. It has potential for onsite diagnosis of R. solani in infected plant samples, seeds, vegetative cuttings and soil samples.

First Report of Elm Canker Caused by Pestalotiopsis mangiferae in the United States

Chinese elm (Ulmus parvifolia Jacq.) is native to China, Korea, and Japan and was introduced to the U.S. to replace the American elm, which is highly susceptible to Dutch elm disease. Cultivar Emmer II trademark Allee elm (or Allee elm) is preferred by nurseries for its rich green foliage and beauty of bark. In the summer of 2011, a new disease was observed on Allee elm at a tree farm in FL. Approximately 1% of elms in the same farm and in residential areas in central Florida had similar canker-like symptoms consisting of tan to orange patches of decomposed and loose bark. These symptoms were observed on the main trunks often extending into branches of affected trees. Cankered sections of the trunk were often several feet in length and penetrated the wood to a depth just under the bark into the phloem. To isolate the causal organism, cankers were gathered from 7 trees and tissues from the margin of 1 to 3 cankers per tree were surface sterilized in 1% sodium hypochlorite, plated on PDA, and incubated at 22°C under a 16-h/8-h light/dark cycle for 7 days. Colonies displayed white, fluffy mycelium with sporadic black acervuli containing aggregated conidia. Conidia were 5-celled with two or more apical appendages or hairs; the three central cells were dark brown and the two outer cells were hyaline (1). Based on conidial morphology, these isolates were putatively identified as Pestalotiopsis spp. To identify the species, the rDNA internal transcribed spacer (ITS) region was sequenced for two field isolates (GenBank Accessions JX999998 and JX999999). A BLASTn search in GenBank revealed 100% identity to Pestalotiopsis mangiferae ITS (JX305704.1). To test Kochs postulates, experiments were performed in the field and greenhouse. A mycelial plug of isolate 11-40 was grown on PDA, inoculated on wounded trunk of healthy 18-month-old Allee elms (n = 48) in the field and 7-month-old Allee elms (n = 12) in the greenhouse. Both experiments were set up as a randomized complete block design. The trunk of each tree was wounded with a scalpel to a depth of 5 mm, the wound was inoculated with a 5-mm2 agar plug from a 7-day PDA culture, and the inoculated wound was wrapped with grafting tape. Plants that served as negative controls [n = 20 (field experiment) and n = 12 (greenhouse)] were mock-inoculated with a sterile PDA plug without mycelial growth. After 4 to 6 months, symptoms consisting of loose or fallen off bark developed on all pathogen-inoculated trees but not on control trees. Control wounds healed with no expansion beyond the original 5 mm inoculation zone, whereas Pestalotiopsis-inoculated cankers expanded to 3 to 8 cm in each direction in 6 months. Pestalotiopsis (confirmed by conidial morphology and ITS sequencing) was reisolated from pathogen-inoculated trees but not from control trees. These experiments were repeated with similar results at least three times, each consisting of 15 replications (greenhouse) and 12 replications (field) with additional Pestalotiopsis isolates. Many other Pestaliopsis spp. have been reported on other shade trees through the U.S. and the world. To our knowledge, this is the first report of P. mangiferae on an Ulmus species in the U.S. and the world. Since elm canker mainly affects the trunk, a featured characteristic of the Allee elm, it can potentially result in economic loss to the ornamental industry. References: (1) Y. Ko et al. Plant Dis. 91:1684, 2007. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Characterization of Phytophthora spp. Isolated from Ornamental Plants in Florida

This report investigates population structure and genetic variability of Phytophthora spp. isolated from botanically diverse plants in Florida. Internal transcribed spacer-based molecular phylogenetic analyses indicate that Phytophthora isolates recovered from ornamental plants in Florida represent a genetically diverse population and that a majority of the isolates belong to Phytophthora nicotianae (73.2%), P. palmivora (18.7%), P. tropicalis (4.9%), P. katsurae (2.4%), and P. cinnamomi (0.8%). Mating type analyses revealed that most isolates were heterothallic, consisting of both mating type A1 (25.2%) and mating type A2 (39.0%), and suggesting that they could outcross. Fungicide sensitivity assays determined that several isolates were moderate to completely insensitive to mefenoxam. In addition, several isolates were also moderately insensitive to additional fungicides with different modes of action. However, correlation analyses did not reveal occurrence of fungicide cross-resistance. These studies suggest that a genetically diverse Phytophthora population infects ornamental crops and the occurrence of mefenoxam-insensitive Phytophthora populations raises concerns about disease management in ornamentals. Mitigating fungicide resistance will require prudent management strategies, including tank mixes and rotation of chemicals with different modes of actions.

First Report of Colletotrichum higginsianum Causing Anthracnose of Arugula (Eruca sativa) in Florida

Arugula (Eruca sativa) is grown in Florida and is an important component in packaged salad products. During spring 2013, leaf lesions on arugula caused significant economic losses in Miami-Dade County, Florida. Symptoms initially appeared as small water-soaked lesions that later became circular, sunken, and white in the center with a dark brown to black halo, up to 4 mm in diameter. Acervuli were found under a dissecting microscope on infected leaf lesions with black spines or setae. Occasionally, small, circular, often longitudinal dark brown spots appeared on leaf branches. Leaf tissues (5 × 5 mm) from lesion margins were surface sterilized in 0.9% sodium hypochlorite for 10 min, rinsed with sterile distilled water, and plated on potato dextrose agar (PDA). PDA plates were incubated at 21°C under 24-h fluorescent lights for 4 to 6 days. The fungus initially produced gray mycelium followed by orange conidial mass. Hyphae of the fungus were septate and hyaline. After 5 to 7 days, the fungus produced acervuli with dark brown to black setae (75 to 130 μm long) (n = 20). Conidia were found in the colonies, which were single celled, oblong, hyaline, and 12 to 25 × 4 to 6 μm (n = 20). The cultural and morphological characteristics of the conidia were similar to those for Colletotrichum higginsianum Sacc (1). To further confirm the species of the isolates, the sequence of the ITS region of rDNA, chitin synthase 1 (CHS1), and actin (ACT) was amplified from isolates 05131 and 05132 using primer pairs ITS 1 and ITS 4 (4), CHS-79F and CHS-354R, and ACT-512F and ACT-783R (3), respectively. The sequenced data of each locus were deposited in GenBank with accessions KF550281.1, KF550282.1, KJ159904, KJ159905, KJ159906, and KJ159907. The resulting sequence of ITS showed 100% identity with sequences of C. higginsianum in JQ005760.1, and sequence of ACT gene showed 100% identity with C. higginsianum in JQ005823.1. The sequence of ACT gene and ITS region had ≤99% identity with other closely related Colletotrichum spp. CHS1 gene had 100% identity with JQ005781.1 belonging to C. higginsianum, and one accession JQ005783.1 belonging to C. fuscum. However, ACT gene and ITS region does not share 100% identity with C. fuscum and therefore, sequence data from three loci proves that isolated pathogen is C. higginsianum. All the above mentioned accessions that shared 100% identity with sequences of isolates used in our study have been previously used to represent the species in the C. destructivum clade in a systematics study (2). To confirm its pathogenicity, a suspension of isolate 05132 at 5 × 105 conidia/ml was sprayed on leaves of five arugula plants until runoff. The other five arugula plants sprayed with water served as non-inoculated controls. Both inoculated and non-inoculated plants were separately covered with a plastic bag to maintain high humidity for 24 h at 27 ± 5°C under natural day/night conditions in the greenhouse. Symptoms first appeared 3 to 4 days after inoculation as small water-soaked lesions, which became sunken with dark brown to black margins. Small circular and longitudinal dark brown spots were also seen on leaf branches as seen initially on naturally infected arugula. No symptoms developed on non-inoculated control plants. C. higginsianum was re-isolated from the lesions with the same morphological characteristics as described above, fulfilling Kochs postulates. To our knowledge, this is the first report of C. higginsianum causing anthracnose of arugula in Florida. This pathogen may potentially affect the salad industry in the United States. References: (1) A. J. Caesar et al. Plant Dis. 94:1166, 2010. (2) P. F. Cannon et al. Stud. Mycol. 73:181, 2012. (3) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Infiltrative and drug-resistant slow-cycling cells support metabolic heterogeneity in glioblastoma

Metabolic reprogramming has been described in rapidly growing tumors, which are thought to mostly contain fast‐cycling cells (FCCs) that have impaired mitochondrial function and rely on aerobic glycolysis. Here, we characterize the metabolic landscape of glioblastoma (GBM) and explore metabolic specificities as targetable vulnerabilities. Our studies highlight the metabolic heterogeneity in GBM, in which FCCs harness aerobic glycolysis, and slow‐cycling cells (SCCs) preferentially utilize mitochondrial oxidative phosphorylation for their functions. SCCs display enhanced invasion and chemoresistance, suggesting their important role in tumor recurrence. SCCs also demonstrate increased lipid contents that are specifically metabolized under glucose‐deprived conditions. Fatty acid transport in SCCs is targetable by pharmacological inhibition or genomic deletion of FABP7, both of which sensitize SCCs to metabolic stress. Furthermore, FABP7 inhibition, whether alone or in combination with glycolysis inhibition, leads to overall increased survival. Our studies reveal the existence of GBM cell subpopulations with distinct metabolic requirements and suggest that FABP7 is central to lipid metabolism in SCCs and that targeting FABP7‐related metabolic pathways is a viable therapeutic strategy.