Development of a real-time PCR assay for detection of hemp russet mite (Aculops cannabicola).

Of the many arthropod species affecting hemp (Cannabis sativa L.) cultivation in the United States, one species of particular importance is the hemp russet mite (Aculops cannabicola, HRM). Hemp russet mite is a microscopic arthropod which feeds on all parts of hemp plants. Due to its minute size, HRM can proliferate undetected for a long time, complicating management efforts and causing serious economic losses. DNA sequencing and PCR assays can facilitate accurate identification and early detection of HRM in infested-plants. Therefore, a real-time SYBR Green based species-specific PCR assay (quantitative PCR, qPCR) was developed for the identification of HRM DNA by amplification of a 104 bp Internal Transcribed Spacer 1 (ITS1) sequence. The detection limit was estimated to be approximately 48 copies of the HRM marker gene sequence. The real-time-PCR assay is rapid, detects all life stages of mite under 2 hours. A 10-fold serial dilution of the plasmid DNA containing the ITS1 insert were used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay showed a strong linear relationship with HRM DNA with R2 of 0.96. The assay was tested against several commonly found hemp pests including two-spotted spider mite and western flower thrips to determine specificity of the assay and to show that no non-target species DNA was amplified. The outcomes of this research will have important applications for agricultural biosecurity through accurate identification of HRM, early detection and timely deployment of management tactics to manage and prevent pest outbreaks.

Assessing the adaptive role of cannabidiol (CBD) in Cannabis sativa defense against cannabis aphids.

Cannabis sativa is known for having unique specialized or secondary metabolites, cannabinoids that are derived from an extension of the terpene pathway in the Cannabis lineage and includes more than 100 other similar metabolites. Despite the assumption that cannabinoids evolved as novel herbivory defense adaptations, there is limited research addressing the role of cannabinoids in C. sativa responses to insect herbivores. Here we investigated the role of cannabidiol (CBD), the predominant cannabinoid in hemp, in plant defense against cannabis aphid (Phorodon cannabis), one of the most damaging pests of hemp. We hypothesize that insect feeding may induce changes in cannabinoids as an adaptive strategy for defense. We found that mean fecundity, net reproductive rate (R0) and adult longevity of cannabis aphids was reduced on the high cannabinoid cultivar compared to the low- cannabinoid cultivar in whole plant assays. In contrast, supplementation of CBD in artificial feeding assays increased aphid fecundity from day 1 to day 3. Additionally, aphid feeding did not impact cannabinoid levels in leaf tissues with the exception of Δ9-tetrahydrocannabinol (THC). This suggests that other cannabinoids and/or metabolites such as terpenes are causing the observed decrease in aphid performance in the whole plant assays. In addition to cannabinoids, C. sativa also possesses a range of defense mechanisms via phytohormone signaling pathways that are well described in other plant species. Indeed, cannabis aphid feeding significantly increased levels of the major phytohormones, salicylic acid, jasmonic acid, and abscisic acid, which are known to be involved in plant defense responses against aphid species. These results highlight the interplay between cannabinoid synthesis and phytohormone pathways and necessitate further investigation into this complex interaction.

Biology and management of hemp russet mite (Acari: Eriophyidae).

Hemp is rapidly becoming a crop of global agricultural importance, and one of the more serious pests of this crop is hemp russet mite (HRM) Aculops cannabicola (Acari: Eriophyidae). Significant knowledge gaps presently exist regarding critical aspects of pest biology, quantification of crop damage, and efficacy of pesticides. Here we assessed the role of cannabidiol (CBD) on HRM performance, efficacy of sulfur treatments in field trials, and effect of hot water immersion with and without surfactants in reducing HRM counts on hemp cuttings. We found that HRM fecundity was reduced on a high-CBD cultivar compared with a low-CBD cultivar in detached leaf assays. In contrast, HRM fecundity and survival were not impacted when reared on high-CBD diet in artificial feeding assays. This suggests that cannabinoids other than CBD may aid in reduction of mite populations on the high-CBD cultivar. Sulfur sprays reduced HRM populations by up to 98% with the greatest effects seen in plants receiving dual applications, one during the vegetative period in July and the second at the initiation of flowering in August. Yields of plants treated with sulfur increased by up to 33%, and there was a further increase in cannabinoid production by up to 45% relative to untreated plants. Hot water immersion treatments with and without surfactant solution reduced HRM on infested hemp cuttings, and no phytotoxicity was observed. This study provides novel approaches to mitigating HRM at multiple stages in hemp production.

Transcriptome analysis of aphid-resistant and susceptible near isogenic lines reveals candidate resistance genes in cowpea (Vigna unguiculata).

BACKGROUND: Cowpea (Vigna unguiculata) is a crucial crop for regions of the world that are prone to both heat and drought; however, the phytotoxic cowpea aphid (Aphis craccivora) impairs plant physiology at low population levels. Both antibiotic and antixenotic forms of resistance to the aphid have been mapped to two quantitative trait loci (QTLs) and near isogenic lines (NILs). The molecular mechanism for this resistance response remains unknown. RESULTS: To understand the genes underlying susceptibility and resistance, two cowpea lines with shared heritage were infested along a time course and characterized for transcriptome variation. Aphids remodeled cowpea development and signaling relative to host plant resistance and the duration of feeding, with resource acquisition and mobilization determining, in part, susceptibility to aphid attack. Major differences between the susceptible and resistant cowpea were identified including two regions of interest housing the most genetic differences between the lines. Candidate genes enabling aphid resistance include both conventional resistance genes (e.g., leucine rich repeat protein kinases) as well as multiple novel genes with no known orthologues. CONCLUSIONS: Our results demonstrate that feeding by the cowpea aphid globally remodels the transcriptome of cowpea, but how this occurs depends on both the duration of feeding and host-plant resistance. Constitutive expression profiles of the resistant genotype link aphid resistance to a finely-tuned resource management strategy that ultimately reduces damage (e.g., chlorosis) and delays cell turnover, while impeding aphid performance. Thus, aphid resistance in cowpea is a complex, multigene response that involves crosstalk between primary and secondary metabolism.

AcDCXR Is a Cowpea Aphid Effector With Putative Roles in Altering Host Immunity and Physiology.

Cowpea, Vigna unguiculata, is a crop that is essential to semiarid areas of the world like Sub-Sahara Africa. Cowpea is highly susceptible to cowpea aphid, Aphis craccivora, infestation that can lead to major yield losses. Aphids feed on their host plant by inserting their hypodermal needlelike flexible stylets into the plant to reach the phloem sap. During feeding, aphids secrete saliva, containing effector proteins, into the plant to disrupt plant immune responses and alter the physiology of the plant to their own advantage. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to identify the salivary proteome of the cowpea aphid. About 150 candidate proteins were identified including diacetyl/L-xylulose reductase (DCXR), a novel enzyme previously unidentified in aphid saliva. DCXR is a member of short-chain dehydrogenases/reductases with dual enzymatic functions in carbohydrate and dicarbonyl metabolism. To assess whether cowpea aphid DCXR (AcDCXR) has similar functions, recombinant AcDCXR was purified and assayed enzymatically. For carbohydrate metabolism, the oxidation of xylitol to xylulose was tested. The dicarbonyl reaction involved the reduction of methylglyoxal, an α-ß-dicarbonyl ketoaldehyde, known as an abiotic and biotic stress response molecule causing cytotoxicity at high concentrations. To assess whether cowpea aphids induce methylglyoxal in plants, we measured methylglyoxal levels in both cowpea and pea (Pisum sativum) plants and found them elevated transiently after aphid infestation. Agrobacterium-mediated transient overexpression of AcDCXR in pea resulted in an increase of cowpea aphid fecundity. Taken together, our results indicate that AcDCXR is an effector with a putative ability to generate additional sources of energy to the aphid and to alter plant defense responses. In addition, this work identified methylglyoxal as a potential novel aphid defense metabolite adding to the known repertoire of plant defenses against aphid pests.

Quantification of Methylglyoxal Levels in Cowpea Leaves in Response to Cowpea Aphid Infestation.

Aphids are a serious pest of crops across the world. Aphids feed by inserting their flexible hypodermal needlelike mouthparts, or stylets, into their host plant tissues. They navigate their way to the phloem where they feed on its sap causing little mechanical damage to the plant. Additionally, while feeding, aphids secrete proteinaceous effectors in their saliva to alter plant metabolism and disrupt plant defenses to gain an advantage over the plant. Even with these arsenals to overcome plant responses, plants have evolved ways to detect and counter with defense responses to curtail aphid infestation. One of such response of cowpea to cowpea aphid infestation, is accumulation of the metabolite methylglyoxal. Methylglyoxal is an α,ß-dicarbonyl ketoaldehyde that is toxic at high concentrations. Methylglyoxal levels increase modestly after exposure to a number of different abiotic and biotic stresses and has been shown to act as an emerging defense signaling molecule at low levels. Here we describe a protocol to measure methylglyoxal in cowpea leaves after cowpea aphid infestation, by utilizing a perchloric acid extraction process. The extracted supernatant was neutralized with potassium carbonate, and methylglyoxal was quantified through its reaction with N-acetyl-L-cysteine to form N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)cysteine, a product that is quantified spectrophotometrically.

Aphid effector Me10 interacts with tomato TFT7, a 14-3-3 isoform involved in aphid resistance.

We demonstrated previously that expression of Macrosiphum euphorbiae salivary protein Me10 enhanced aphid reproduction on its host tomato (Solanum lycopersicum). However, the mechanism of action of Me10 remained elusive. To confirm the secretion of Me10 by the aphid into plant tissues, we produced Me10 polyclonal antibodies. To identify the plant targets of Me10, we developed a tomato immune induced complementary DNA yeast two-hybrid library and screened it with Me10 as bait. Immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays were performed to validate one of the interactions in planta, and virus-induced gene silencing was used for functional characterization in tomato. We demonstrated that Me10 is secreted into the plant tissues and interacts with tomato 14-3-3 isoform 7 (TFT7) in yeast. Immunoprecipitation assays confirmed that Me10 and its homologue in Aphis gossypii, Ag10k, interact with TFT7 in planta. Further, BiFC revealed that Me10 interaction with TFT7 occurs in the plant cell cytoplasm. While silencing of TFT7 in tomato leaves did not affect tomato susceptibility to M. euphorbiae, it enhanced longevity and fecundity of A. gossypii, the non-host aphid. Our results suggest the model whereby TFT7 plays a role in aphid resistance in tomato and effectors of the Me10/Ag10k family interfere with TFT7 function during aphid infestation.

Sequence analysis of the potato aphid Macrosiphum euphorbiae transcriptome identified two new viruses.

The potato aphid, Macrosiphum euphorbiae, is an important agricultural pest that causes economic losses to potato and tomato production. To establish the transcriptome for this aphid, RNA-Seq libraries constructed from aphids maintained on tomato plants were used in Illumina sequencing generating 52.6 million 75-105 bp paired-end reads. The reads were assembled using Velvet/Oases software with SEED preprocessing resulting in 22,137 contigs with an N50 value of 2,003bp. After removal of contigs from tomato host origin, 20,254 contigs were annotated using BLASTx searches against the non-redundant protein database from the National Center for Biotechnology Information (NCBI) as well as IntereProScan. This identified matches for 74% of the potato aphid contigs. The highest ranking hits for over 12,700 contigs were against the related pea aphid, Acyrthosiphon pisum. Gene Ontology (GO) was used to classify the identified M. euphorbiae contigs into biological process, cellular component and molecular function. Among the contigs, sequences of microbial origin were identified. Sixty five contigs were from the aphid bacterial obligate endosymbiont Buchnera aphidicola origin and two contigs had amino acid similarities to viruses. The latter two were named Macrosiphum euphorbiae virus 2 (MeV-2) and Macrosiphum euphorbiae virus 3 (MeV-3). The highest sequence identity to MeV-2 had the Dysaphis plantaginea densovirus, while to MeV-3 is the Hubei sobemo-like virus 49. Characterization of MeV-2 and MeV-3 indicated that both are transmitted vertically from adult aphids to nymphs. MeV-2 peptides were detected in the aphid saliva and only MeV-2 and not MeV-3 nucleic acids were detected inside tomato leaves exposed to virus-infected aphids. However, MeV-2 nucleic acids did not persist in tomato leaf tissues, after clearing the plants from aphids, indicating that MeV-2 is likely an aphid virus.

Hyperphosphatemia, hypocalcemia and increased serum potassium concentration as distinctive features of early hypomagnesemia in magnesium-deprived mice.

Magnesium-deficient patients show dysfunctional calcium (Ca(2+)) metabolism due to defective parathyroid hormone (PTH) secretion. In mice and rats, long-term magnesium (Mg(2+)) deprivation causes hyperphosphaturia and increases fibroblast growth factor 23 (FGF23) secretion, despite normal serum phosphate (Pi) and Ca(2+). Electrolyte disturbances during early hypomagnesemia may explain the response of mice to long-term Mg(2+) deprivation, but our knowledge of electrolyte homeostasis during this stage is limited. This study compares the effect of both short- and long-term Mg(2+) restriction on the electrolyte balance in mice. Mice were fed control or Mg(2+)-deficient diets for one to three days, one week, or three weeks. Prior to killing the mice, urine was collected over 24 h using metabolic cages. Within 24 h of Mg(2+) deprivation, hypomagnesemia, hypocalcemia and hyperphosphatemia developed, and after three days of Mg(2+) deprivation, serum potassium (K(+)) was increased. These changes were accompanied by a reduction in urinary volume, hyperphosphaturia, hypocalciuria and decreased Mg(2+), sodium (Na(+)) and K(+) excretion. Surprisingly, after one week of Mg(2+) deprivation, serum K(+), Pi and Ca(2+) had normalized, showing that mineral homeostasis is most affected during early hypomagnesemia. Serum Pi and K(+) are known to stimulate secretion of FGF23 and aldosterone, which are usually elevated during Mg(2+) deficiency. Thus, the hyperphosphatemia and increased serum K(+) concentration observed during short-term Mg(2+) deprivation may help our understanding of adaptation to chronic Mg(2+) deficiency.