Identification of Cold Tolerance Transcriptional Regulatory Genes in Seedlings of Medicago sativa L. and Medicago falcata L.

Alfalfa species Medicago sativa L. (MS) and Medicago falcata L. (MF), globally prominent perennial leguminous forages, hold substantial economic value. However, our comprehension of the molecular mechanisms governing their resistance to cold stress remains limited. To address this knowledge gap, we scrutinized and compared MS and MF cold-stress responses at the molecular level following 24 h and 120 h low-temperature exposure (4 °C). Our study revealed that MF had superior physiological resilience to cold stress compared with MS, and its morphology was healthier under cold stress, and its malondialdehyde content and superoxide dismutase activity increased, first, and then decreased, while the soluble sugar content continued to accumulate. Transcriptome analysis showed that after 120 h of exposure, there were different gene-expression patterns between MS and MF, including 1274 and 2983 genes that were continuously up-regulated, respectively, and a total of 923 genes were included, including star cold-resistant genes such as ICE1 and SIP1. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed numerous inter-species differences in sustained cold-stress responses. Notably, MS-exclusive genes included a single transcription factor (TF) gene and several genes associated with a single DNA repair-related pathway, whereas MF-exclusive genes comprised nine TF genes and genes associated with 14 pathways. Both species exhibited high-level expression of genes encoding TFs belonging to AP2-EREBP, ARR-B, and bHLH TF families, indicating their potential roles in sustaining cold resistance in alfalfa-related species. These findings provide insights into the molecular mechanisms governing cold-stress responses in MS and MF, which could inform breeding programs aimed at enhancing cold-stress resistance in alfalfa cultivars.

Comparative transcriptome analysis and meta-QTLs mapping reveal the regulatory mechanism of cold tolerance in rice at the budding stage.

Rice (Oryza sativa L.) is one of the most extensively farmed food crops, but its development and productivity are significantly impacted by cold stress during the budding period. In this study, transcriptome sequencing was conducted on two types of rice: the cold-sensitive indica rice A117 and the substantially cold-tolerant japonica rice B106 under control and cold treatments. Differentially expressed genes between the two materials under cold conditions were analyzed using GO and KEGG enrichment analyses. The results revealed that processes such as the TCA cycle, glycolysis/glycogenesis, oxidative phosphorylation, and glutathione metabolism contribute to B106's cold tolerance. Additionally, an enrichment analysis of cold-induced genes in each material and shared genes identified significant enrichment in pathways such as glutathione metabolism, phenylpropanoid biosynthesis, and photosynthesis-antenna proteins. Initial cold tolerance QTLs at the rice bud stage were collected from published literature, and meta-QTL mapping identified 9 MQTLs. Gene expression profiling led to the identification of 75 potential DEGs within the 9 MQTLs region, from which four candidate genes (Os02g0194100, Os03g0802500, Os05g0129000, and Os07g0462000) were selected using qRT-PCR and gene annotation. These findings provide genetic resources for further research on the molecular mechanisms underlying rice's response to cold stress during the bud stage.

Enhancement of Thermal Tolerance and Growth Performances of Asian Seabass (Lates calcarifer) Fed with Grape Extract Supplemented Feed.

Cold snaps during the winter present a critical challenge for Asian seabass (Lates calcarifer) in Taiwan, as sudden temperature drops significantly affect their growth and survival. This study explores the effects of dietary grape extract (GE) from Vitis vinifera on the growth performance, oxidative stress regulation, and thermal tolerance of this commercially valuable fish. Over a 60-day feeding trial, four dietary groups were tested: a control diet without GE and three diets supplemented with GE at 2% (GE20), 3% (GE30), and 4% (GE40) with commercial feed. The results demonstrated that GE supplementation positively influenced growth, with the GE20 group achieving the best weight gain and feed conversion ratio among all groups. The upregulation of the growth-related gene igf-1 in the liver of the GE20 group further supported its superior growth performance. Additionally, GE-fed groups showed increased expression of antioxidant-related genes sod1 and sod2 in the liver, while gpx1 exhibited a significant increase only in the GE20 group, indicating enhanced antioxidant defenses. Cat gene expression remained unchanged, and higher GE doses reduced the expression of gpx1, cat, and igf-1. Furthermore, GE supplementation improved cold tolerance in all treated groups compared to the control. These findings suggest that dietary GE at 20 g/kg is particularly effective in enhancing growth performance and cold tolerance in Asian seabass, offering a promising strategy for boosting fish health and adaptability in aquaculture.

Flavonoid Synthesis Pathway Response to Low-Temperature Stress in a Desert Medicinal Plant, Agriophyllum Squarrosum (Sandrice).

Background/Objectives:Agriophyllum squarrosum (L.) Moq. (A. squarrosum), also known as sandrice, is an important medicinal plant widely distributed in dunes across all the deserts of China. Common garden trials have shown content variations in flavonoids among the ecotypes of sandrice, which correlated with temperature heterogeneity in situ. However, there have not been any environmental control experiments to further elucidate whether the accumulation of flavonoids was triggered by cold stress; Methods: This study conducted a four-day ambient 4 °C low-temperature treatment on three ecotypes along with an in situ annual mean temperature gradient (Dulan (DL), Aerxiang (AEX), and Dengkou (DK)); Results: Target metabolomics showed that 12 out of 14 flavonoids in sandrice were driven by cold stress. Among them, several flavonoids were significantly up-regulated, such as naringenin and naringenin chalcone in all three ecotypes; isorhamnetin, quercetin, dihydroquercetin, and kaempferol in DL and AEX; and astragalin in DK. They were accompanied by 19 structural genes of flavonoid synthesis and 33 transcription factors were markedly triggered by cold stress in sandrice. The upstream genes, AsqAEX006535-CHS, AsqAEX016074-C4H, and AsqAEX004011-4CL, were highly correlated with the enrichment of naringenin, which could be fine-tuned by AsqAEX015868-bHLH62, AsqAEX001711-MYB12, and AsqAEX002220-MYB1R1; Conclusions: This study sheds light on how desert plants like sandrice adapt to cold stress by relying on a unique flavonoid biosynthesis mechanism that regulating the accumulation of naringenin. It also supports the precise development of sandrice for the medicinal industry. Specifically, quercetin and isorhamnetin should be targeted for development in DL and AEX, while astragalin should be precisely developed in DK.

OsWRKY70 Plays Opposite Roles in Blast Resistance and Cold Stress Tolerance in Rice.

The transcription factor WRKYs play pivotal roles in the adapting to adverse environments in plants. Prior research has demonstrated the involvement of OsWRKY70 in resistance against herbivores and its response to abiotic stress. Here, we reported the functional analysis of OsWRKY70 in immunity against fungal diseases and cold tolerance. The results revealed that OsWRKY70 was induced by various Magnaporthe oryzae strains. Knock out mutants of OsWRKY70, which were generated by the CRISPR/Cas9 system, exhibited enhanced resistance to M. oryzae. This was consistent with fortifying the reactive oxygen species (ROS) burst after inoculation in the mutants, elevated transcript levels of defense-responsive genes (OsPR1b, OsPBZ1, OsPOX8.1 and OsPOX22.3) and the observation of the sluggish growth of invasive hyphae under fluorescence microscope. RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) validations demonstrated that differentially expressed genes were related to plant-pathogen interactions, hormone transduction and MAPK cascades. Notably, OsbHLH6, a key component of the JA signaling pathway, was down-regulated in the mutants compared to wild type plants. Further investigation confirmed that OsWRKY70 bound to the promoter of OsbHLH6 by semi-in vivo chromatin immunoprecipitation (ChIP). Additionally, the loss-function of OsWRKY70 impaired cold tolerance in rice. The enhanced susceptibility in the mutants characterized by excessive ROS production, elevated ion leakage rate and increased malondialdehyde content, as well as decreased activity of catalase (CAT) and peroxidase (POD) under low temperature stress was, which might be attributed to down-regulation of cold-responsive genes (OsLti6b and OsICE1). In conclusion, our findings indicate that OsWRKY70 negatively contributes to blast resistance but positively regulates cold tolerance in rice, providing a strategy for crop breeding with tolerance to stress.

The seed endophytic microbe Microbacterium testaceum M15 enhances the cold tolerance and growth of rice (Oryza sativa L.).

The potential of seed endophytic microbes to enhance plant growth and resilience is well recognized, yet their role in alleviating cold stress in rice remains underexplored due to the complexity of these microbial communities. In this study, we investigated the diversity of seed endophytic microbes in two rice varieties, the cold-sensitive CB9 and the cold-tolerant JG117. Our results revealed significant differences in the abundance of Microbacteriaceae, with JG117 exhibiting a higher abundance under both cold stress and room temperature conditions compared to CB9. Further analysis led to the identification of a specific cold-tolerant microbe, Microbacterium testaceum M15, in JG117 seeds. M15-inoculated CB9 plants showed enhanced growth and cold tolerance, with a germination rate increase from 40 % to 56.67 % at 14℃ and a survival rate under cold stress (4℃) doubling from 22.67 % to 66.67 %. Additionally, M15 significantly boosted chlorophyll content by over 30 %, increased total protein by 16.31 %, reduced malondialdehyde (MDA) levels by 37.76 %, and increased catalase activity by 26.15 %. Overall, our study highlights the potential of beneficial endophytic microbes like M. testaceum M15 in improving cold tolerance in rice, which could have implications for sustainable agricultural practices and increased crop productivity in cold-prone regions.

SlBTB19 interacts with SlWRKY2 to suppress cold tolerance in tomato via the CBF pathway.

Cold stress restricts the metabolic and physiological activities of plants, thereby affecting their growth and development. Although broad-complex, tramtrack, and bric-à-brac (BTB) proteins are essential for diverse biological processes and stress responses, the mechanisms underlying BTB-mediated cold responses remain not fully understood. Here, we characterize the function of the cold-induced SlBTB19 protein in tomato (Solanum lycopersicum). Overexpression of SlBTB19 resulted in increased plant sensitivity to cold stress, whereas SlBTB19 knockout mutants exhibited a cold-tolerance phenotype. Further analyses, including protein-protein interaction studies and cell-free degradation assays, revealed that SlBTB19 interacts with and destabilizes the transcription factor SlWRKY2. Using virus-induced gene silencing (VIGS) to silence SlWRKY2 in both wild-type and slbtb19 mutants, we provided genetic evidence that SlWRKY2 acts downstream of SlBTB19 in regulating cold tolerance. Importantly, we demonstrated that SlWRKY2 positively regulates cold tolerance in a CRT/DRE binding factor (CBF)-dependent manner. Under cold stress, SlWRKY2 binds to the W-box in the CBF1 and CBF3 promoters, directly activating their expression. In summary, our findings identify a SlBTB19-SlWRKY2 module that negatively regulates the CBF-dependent cold tolerance pathway in tomato.

Cold Tolerance in the Nematode Caenorhabditis elegans.

Organisms receive environmental information and respond accordingly in order to survive and proliferate. Temperature is the environmental factor of most immediate importance, as exceeding its life-supporting range renders essential biochemical reactions impossible. In this chapter, we introduce the mechanisms underlying cold tolerance and temperature acclimation in a model organism-the nematode Caenorhabditis elegans, at molecular and physiological levels. Recent investigations utilizing molecular genetics and neural calcium imaging have unveiled a novel perspective on cold tolerance within the nematode worm. Notably, the ASJ neuron, previously known to possess photosensitive properties, has been found to sense temperature and regulate the sperm and gut cell-mediated pathway underlying cold tolerance. We will also explore C. elegans' cold tolerance and cold acclimation at the molecular and tissue levels.

Overwintering potential of the Mediterranean fruit fly (Diptera: Tephritidae) in Austria.

The Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann), one of the most important invasive pests of fresh fruits and vegetables from the coastal Mediterranean habitats, is expanding its current geographic distribution to cooler more temperate areas of Europe. Every year since 2010 the fly is detected in the area of Vienna, Austria. However, whether it can establish permanent populations is not known. In this current paper, the capacity of C. capitata to overwinter in Vienna, Austria (48.1° northern latitude) was studied over 2 consecutive winter seasons (2020-2022). Overwintering trials with different life stages (larva, pupa, and adult) of C. capitata were performed in the open field and in the protected environment of a basement without a heating system. Control flies were kept under constant conditions in a climate chamber (25 °C, 60% RH, 14:10 L:D). Our data showed that no life stage of the Mediterranean fruit fly was able to survive the Austrian winter in the open field. However, in the protected environment C. capitata outlived the winter months in all studied life stages at least in small numbers and several surviving females were able to lay eggs at the time of the following fruiting season. Implications of these findings for the ongoing geographic range expansion of the pest in temperate European countries are discussed.

HOS15-mediated turnover of PRR7 enhances freezing tolerance.

Arabidopsis PSEUDORESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome-dependent degradation to discretely phase maximal expression in early evening. While its repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 abundance is unknown. We used mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein-protein interactions at low temperatures. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP-qPCR at C-REPEAT BINDING FACTOR1 (CBF1) and COLD-REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants. HOS15 mediates PRR7 turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoters of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity. We propose a novel mechanism whereby HOS15-mediated degradation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways that lead to increased freezing tolerance.

Innovative Postharvest Management for Hass Avocado at the Preclimacteric Stage: A Combined Technology with GABA and 1-MCP.

Avocado (Persea americana Mill.) is a subtropical climacteric fruit with a limited shelf life due to its high sensitivity to low temperatures. Chilling injury (CI) produced by cold storage displays symptoms in avocado fruit such as irregular ripening, darkening of the mesocarp, hardening of vascular strands, lipid oxidation with "off flavors", and pitting and darkening of the skin, increasing weight loss. Accordingly, we studied the effect of γ-aminobutyric acid (GABA) and 1-methylcyclopropene (1-MCP) alone or in combination as postharvest treatments to maintain quality and to increase cold tolerance. Hass avocados were stored at 5 °C plus 5 days at room temperature. The results showed that the combined treatment improved fruit quality parameters as compared with control fruit and with those treated with only 1-MCP or GABA. The combined treatment delayed synergistically the postharvest ripening process. This delayed pattern was concomitant with a delayed ethylene pattern in GABA + 1-MCP or 1-MCP fruit batches. CI symptoms and electrolyte leakage were minimized in all GABA and 1-MCP fruit batches specifically in the combined treatment. For this reason, the synergistic effect of the combination of treatments may be recommended as an effective alternative strategy to prolong the postharvest quality of avocado during refrigerated storage.

Genetic variation in a heat shock transcription factor modulates cold tolerance in maize.

Understanding how maize (Zea mays) responds to cold stress is crucial for facilitating breeding programs of cold-tolerant varieties. Despite extensive utilization of the genome-wide association study (GWAS) approach for exploring favorable natural alleles associated with maize cold tolerance, few studies have successfully identified candidate genes that contribute to maize cold tolerance. In this study, we used a diverse panel of inbred maize lines collected from different germplasm sources to perform a GWAS on variations in the relative injured area of maize true leaves during cold stress-a trait very closely correlated with maize cold tolerance. We identified HSF21, which encodes a B-class heat shock transcription factor (HSF) that positively regulates cold tolerance at both the seedling and germination stages. Natural variations in the promoter of the cold-tolerant HSF21Hap1 allele led to increased HSF21 expression under cold stress by inhibiting binding of the basic leucine zipper bZIP68 transcription factor, a negative regulator of cold tolerance. By integrating transcriptome deep sequencing, DNA affinity purification sequencing, and targeted lipidomic analysis, we revealed the function of HSF21 in regulating lipid metabolism homeostasis to modulate cold tolerance in maize. In addition, we found that HSF21 confers maize cold tolerance without incurring yield penalties. Collectively, this study establishes HSF21 as a key regulator that enhances cold tolerance in maize, providing valuable genetic resources for breeding of cold-tolerant maize varieties.

A novel strategy of artificially regulating plant rhizosphere microbial community to promote plant tolerance to cold stress.

Artificial regulation of plant rhizosphere microbial communities through the synthesis of microbial communities is one of the effective ways to improve plant stress resistance. However, the process of synthesizing stress resistant microbial communities with excellent performance is complex, time-consuming, and costly. To address this issue, we proposed a novel strategy for preparing functional microbial communities. We isolated a cultivable cold tolerant bacterial community (PRCBC) from the rhizosphere of peas, and studied its effectiveness in assisting rice to resist stress. The results indicate that PRCBC can not only improve the ability of rice to resist cold stress, but also promote the increase of rice yield after cold stress relieved. This is partly because PRCBC increases the nitrogen content in the rhizosphere soil, and promotes rice's absorption of nitrogen elements, thereby promoting rice growth and enhancing its ability to resist osmotic stress. More importantly, the application of PRCBC drives the succession of rice rhizosphere microbial communities, and promotes the succession of rice rhizosphere microbial communities towards stress resistance. Surprisingly, PRCBC drives the succession of rice rhizosphere microbial communities towards a composition similar to PRCBC. This provides a feasible novel method for artificially and directionally driving microbial succession. In summary, we not only proposed a novel and efficient strategy for preparing stress resistant microbial communities to promote plant stress resistance, but also unexpectedly discovered a possible directionally driving method for soil microbial community succession.

Chlorogenic Acid as a Promising Tool for Mitigating Chilling Injury: Cold Tolerance and the Ripening Effect on Tomato Fruit (Solanum lycopersicum L.).

Tomato fruit (Solanum lycopersicum L.) has a very brief storability, displaying chilling injury (CI) when stored in cold conditions used to delay ripening. For this reason, in this study, different concentrations (10, 50, and 100 mg L-1) of chlorogenic acid (ChA) were assayed to evaluate its effectiveness in maintaining fruit quality traits and mitigating CI symptoms in tomatoes. Our results showed that ChA treatments effectively delayed weight loss and maintained fruit firmness, with optimal results observed at 50 mg L-1. In general, higher concentrations did not result in significant quality improvements. Additionally, ChA-treated tomatoes exhibited reduced values in malondialdehyde (MDA) content and electrolyte leakage (EL), indicating improved membrane integrity and reduced oxidative damage. ChA treatments also maintained a higher total phenolic content (TPC) during storage, with significant levels of individual polyphenols such as rutin, neochlorogenic acid, and p-coumaric acid, suggesting enhanced antioxidant capacity and better preservation of fruit quality. This is the first time the potential of ChA to reduce CI has been evaluated in any fruit species, and its impact in tomato ripening is shown to uphold fruit quality during cold storage, prolonging the storability of tomatoes. In particular, we highlight its natural origin and effectiveness as a postharvest treatment.

Experimental evidence of a Neotropical pest insect moderately tolerant to complete freezing.

Due to climate change, many regions are experiencing progressively milder winters. Consequently, pest insects from warm regions, particularly those with some tolerance to low temperatures, could expand their geographic range into these traditionally colder regions. The palm borer moth (Paysandisia archon) is a Neotropical insect that in recent decades has reached Europe and Asia as one of the worst pests of palm trees. Little is known about its ability to tolerate moderately cold winters and, therefore, to colonize new areas. In this work, we characterized the cold tolerance of Paysandisia archon by measuring its thermal limits: median lethal-temperature, LT50, chill-coma onset temperature, CTmin, supercooling point, SCP, freezing time and freezing survival. We found that this species was able to survive short periods of complete freezing, with survival rates of 87% after a 30-min freezing exposure, and 33% for a 1 h-exposure. It is then a moderately freeze-tolerant species, in contrast to all other lepidopterans native to warm areas, which are freeze-intolerant. Additionally, we investigated whether this insect improved its cold tolerance after either short or long pre-exposure to sub-lethal low temperatures. To that end, we studied potential changes in the main thermo-tolerance parameters and, using X-ray Computed Tomography, also in the morphological components of pretreated animals. We found that short pre-exposures did not imply significant changes in the SCP and CTmin values. In contrast, larvae with long pretreatments improved their survival to both freezing and low temperatures, and required longer times for complete freezing than the other groups. These long-term pre-exposed larvae also presented several morphological changes, including a reduction in water content that probably explained, at least in part, their longer freezing time and higher freezing survival. Our results represent the first cold tolerance characterization of this pest insect, which could be relevant to better design strategies to combat it.

The intron binding protein EMB-4 is an opposite regulator of cold and high temperature tolerance in Caenorhabditis elegans.

Adaptation and tolerance to changes in heat and cold temperature are essential for survival and proliferation in plants and animals. However, there is no clear information regarding the common molecules between animals and plants. In this study, we found that heat, and cold tolerance of the nematode Caenorhabditis elegans is oppositely regulated by the RNA-binding protein EMB-4, whose plant homolog contains polymorphism causing heat tolerance diversity. Caenorhabditis elegans alters its cold and heat tolerance depending on the previous cultivation temperature, wherein EMB-4 respectively acts as a positive and negative controller of heat and cold tolerance by altering gene expression. Among the genes whose expression is regulated by EMB-4, a phospholipid scramblase, and an acid sphingomyelinase, which are involved in membrane lipid metabolism, were found to play essential roles in the negative regulation of heat tolerance.

HOS15-mediated turnover of PRR7 enhances freezing tolerance.

Arabidopsis PSEUDO RESPONSE REGULATOR7 (PRR7) is a core component of the circadian oscillator which also plays a crucial role in freezing tolerance. PRR7 undergoes proteasome-dependent degradation to discretely phase maximal expression in early evening. While its transcriptional repressive activity on downstream genes is integral to cold regulation, the mechanism of the conditional regulation of the PRR7 protein activity is unknown. We used double mutant analysis, protein interaction and ubiquitylation assays to establish that the ubiquitin ligase adaptor, HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 15 (HOS15), controls the protein accumulation pattern of PRR7 through direct protein-protein interactions. Freezing tolerance and electrolyte leakage assays show that PRR7 enhances cold temperature sensitivity, supported by ChIP-qPCR at C-REPEAT BINDING FACTOR (CBF) and COLD REGULATED 15A (COR15A) promoters where PRR7 levels were higher in hos15 mutants. We establish that HOS15 mediates PRR7 protein turnover through enhanced ubiquitylation at low temperature in the dark. Under the same conditions, increased PRR7 association with the promoter regions of CBFs and COR15A in hos15 correlates with decreased CBF1 and COR15A transcription and enhanced freezing sensitivity. We propose a novel mechanism whereby HOS15-mediated regulation of PRR7 provides an intersection between the circadian system and other cold acclimation pathways leading to freezing tolerance through upregulation of CBF1 and COR15A.

Integrated Transcriptomic and Proteomic Analysis Reveals Molecular Mechanisms of the Cold Stress Response during the Overwintering Period in Blueberries (Vaccinium spp.).

In China, the Liaodong Peninsula is an important growing area for blueberries because of the high organic matter content in the soil, the abundance of light, and the large temperature difference between day and night. However, the low temperature and relative humidity of the air during the winter and early spring in the Liaodong Peninsula are the main reasons for the damage to blueberry plants. Here, we documented the transcriptome and proteome dynamics in response to cold stress in three blueberry cultivars ('Northland', 'Bluecrop', and 'Berkeley'). Functional enrichment analysis indicated that many differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) were mainly involved in the pathways of protein processing in the endoplasmic reticulum, the glutathione metabolism pathway, and ribosomes. We identified 12,747 transcription factors (TFs) distributed in 20 families. Based on our findings, we speculated that cold tolerance development was caused by the expression of calcium-related genes (CDPKs and CMLs), glutathione proteins, and TFs (NAC, WRKY, and ERF). Our investigation found that three cultivars experienced cold damage when exposed to temperatures between -9 °C and -15 °C in the field. Therefore, the cold resistance of blueberries during overwintering should not only resist the influence of low temperatures but also complex environmental factors such as strong winds and low relative humidity in the air. The order of cold resistance strength in the three blueberry cultivars was 'Berkeley', 'Bluecrop', and 'Northland'. These results provide a comprehensive profile of the response to cold stress, which has the potential to be used as a selection marker for programs to improve cold tolerance in blueberries.

Effect of a severe cold spell on overwintering survival of an invasive forest insect pest.

Cold temperatures can play a significant role in the range and impact of pest insects. Severe cold events can reduce the size of insect outbreaks and perhaps even cause outbreaks to end. Measuring the precise impact of cold events, however, can be difficult because estimates of insect mortality are often made at the end of the winter season. In late January 2023 long-term climate models predicted a significant cold event to occur over eastern North America. We used this event to evaluate the immediate impact on hemlock woolly adelgid (Adelges tsugae Annand) overwintering mortality at four sites on the northern edge of the insects invaded range in eastern North America. We observed complete mortality, partial mortality and no effects on hemlock woolly adelgid mortality that correlated with the location of populations and strength of the cold event. Our data showed support for preconditioning of overwintering adelgids having an impact on their overwintering survival following this severe cold event. Finally, we compared the climatic conditions at our sites to historical weather data and previous observations of mortality in Nova Scotia. The cold event observed in February 2023 resulted in the coldest temperatures observed at these sites, including the period within which hemlock woolly adelgid invaded, suggesting cold conditions, especially under anthropogenic climate forcing, may not be a limiting factor in determining the ultimate northern range of hemlock woolly adelgid in eastern North America.