Greg McCollum

Citrus, like many other tropical and subtropical fruit are sensitive to chilling temperatures. However, application of a pre-storage temperature conditioning (CD) treatment at 16°C for 7 d or of a hot water brushing (HWB) treatment at 60°C for 20 sec remarkably enhances chilling tolerance and reduces the development of chilling injuries (CI) upon storage at 5°C. In the current research, we proposed to identify and characterize grapefruit genes that are induced by CD, and may contribute to the acquisition of fruit chilling tolerance, by two different molecular approaches: cDNA array analysis and PCR cDNA subtraction. In addition, following the recent development and commercialization of the new Affymetrix Citrus Genome Array, we further performed genome-wide transcript profiling analysis following exposure to CD and chilling treatments. To conduct the cDNA array analysis, we constructed cDNA libraries from the peel tissue of CD- and HWB-treated grapefruit, and performed an EST sequencing project including sequencing of 3,456 cDNAs from each library. Based on the obtained sequence information, we chose 70 stress-responsive and chilling-related genes and spotted them on nylon membranes. Following hybridization the constructed cDNA arrays with RNA probes from control and CD-treated fruit and detailed confirmations by RT-PCR analysis, we found that six genes: lipid-transfer protein, metallothionein-like protein, catalase, GTP-binding protein, Lea5, and stress-responsive zinc finger protein, showed higher transcript levels in flavedo of conditioned than in non-conditioned fruit stored at 5 ᵒC. The transcript levels of another four genes: galactinol synthase, ACC oxidase, temperature-induced lipocalin, and chilling-inducible oxygenase, increased only in control untreated fruit but not in chilling-tolerant CD-treated fruit.  By PCR cDNA subtraction analysis we identified 17 new chilling-responsive and HWB- and CD-induced genes. Overall, characterization of the expression patterns of these genes as well as of 11 more stress-related genes by RNA gel blot hybridizations revealed that the HWB treatment activated mainly the expression of stress-related genes(HSP19-I, HSP19-II, dehydrin, universal stress protein, EIN2, 1,3;4-β-D-glucanase, and SOD), whereas the CD treatment activated mainly the expression of lipid modification enzymes, including fatty acid disaturase2 (FAD2) and lipid transfer protein (LTP). Genome wide transcriptional profiling analysis using the newly developed Affymetrix Citrus GeneChip® microarray (including 30,171 citrus probe sets) revealed the identification of three different chilling-related regulons: 1,345 probe sets were significantly affected by chilling in both control and CD-treated fruits (chilling-response regulon), 509 probe sets were unique to the CD-treated fruits (chilling tolerance regulon), and 417 probe sets were unique to the chilling-sensitive control fruits (chilling stress regulon). Overall, exposure to chilling led to expression governed arrest of general cellular metabolic activity, including concretive down-regulation of cell wall, pathogen defense, photosynthesis, respiration, and protein, nucleic acid and secondary metabolism. On the other hand, chilling enhanced various adaptation processes, such as changes in the expression levels of transcripts related to membranes, lipid, sterol and carbohydrate metabolism, stress stimuli, hormone biosynthesis, and modifications in DNA binding and transcription factors.  

The devastation to the US citrus industries from huanglongbing (HLB or citrus greening disease, caused by 'Candidatus Liberibacter asiaticus') is spread during feeding by the Asian citrus psyllid (ACP) Diaphorina citri. Innovative echnologies, such as RNA suppression by RNAi, morpholino oligos, or gene editing tools, including CRISPR/Cas9, all provide non-transgenic strategies, as well as transgenic solutions to manage arthropod vectors, pests and pathogens. Current results from researchers report that these methods enable suppression of ACP vectors, their endosymbionts and the Liberibacter pathogens in infected citrus trees. This chapter reviews several emerging technologies and strategies that enable direct targeting of pathogenic microbes including bacteria in citrus trees, and the critical genes specific to arthropod vectors, such as ACP and their endosymbionts. RNAi-based approaches continue to make advances to improve persistence and activity, especially when applied a...

Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens , a culturable surrogate for the unculturable HLB-associated bacterium “ Candidatus Liberibacter asiaticus,” we tested the hypothesis that members of the citrus microbiome produce potential anti-“ Ca . Liberibacter asiaticus” natural products with potential anti-“ Ca . Liberibacter asiaticus” activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens .

Huanglongbing (HLB) disease is the most serious threat to citrus production worldwide and, in the last decade, has devastated the Florida citrus industry. In the United States, HLB is associated with the phloem-limited α-proteobacterium ‘Candidatus Liberibacter asiaticus’ and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri). Significant effort is being put forth to develop novel citrus germplasm that has a lower propensity to succumb to HLB than do currently available varieties. Effective methods of screening citrus germplasm for susceptibility to HLB are essential. In this study, we exposed small, grafted trees of 16 citrus types to free-ranging ACP vectors and ‘Ca. L. asiaticus’ inoculum in the greenhouse. During 45 weeks of exposure to ACP, the cumulative incidence of ‘Ca. L. asiaticus’ infection was 70%. Trees of Citrus macrophylla and C. medica were most susceptible to ‘Ca. L. asiaticus’, with 100% infection by the end of the test period in three trials, whil...

ABSTRACTA pre‐storage conditioning (CD) treatment of 16 °C for 7 d enhanced chilling tolerance of grapefruit and reduced the development of chilling injuries during storage at 5 °C. To gain a better understanding of the molecular mechanisms involved in the responses of citrus fruit to low temperatures, we performed genome‐wide transcriptional profiling analysis of RNA isolated from grapefruit flavedo using the newly developed Affymetrix Citrus GeneChip microarray. Utilizing very restrictive cut‐off criteria, including pair‐wise anova comparisons significantly different at P ≤ 0.05 and induction or repression of transcript levels by at least fourfold, we found that out of 30 171 probe sets on the microarray, 1345 probe sets were significantly affected by chilling in both control and CD‐treated fruits, 509 probe sets were affected by chilling specifically in the CD‐treated fruits, and 417 probe sets were specifically expressed in chilling‐sensitive control fruits. Overall, exposure to...

Because agrochemicals and conventional breeding for disease resistance have not been sufficient in controlling huanglongbing (HLB), alternative strategies for sustaining citrus crops have attracted attention. Since no HLB resistance has been identified within cultivated citrus, transgenic solutions to the disease have become a focus of breeding programs. Among these solutions, use of antimicrobial peptides (AMPs) to enhance plant resistance is at the forefront. Virtually all organisms possess an innate immune response involving AMPs that can counter a microbial infection. At the USHRL, we have chosen to use synthetic or plant AMPs over those of animal origin. The first of the AMPs selected was D4E1, a 17 amino acid synthetic AMP which forms a β sheet (Lucca et al., 1998) and is active against Agrobacterium tumefaciens in poplar (Mentag et al., 2003). It is a highly active AMP with a minimum inhibitory concentration (MIC) less than 1 μM (Stover et al., 2008). We constructed a binary ...

Many recent publications and considerable discussion have focused on root health and the importance of rootstock response on huanglongbing (HLB) development, with special emphasis on bicarbonate intolerance of trifoliate hybrid rootstocks affecting HLB severity. USDA advanced citrus scion selections are routinely planted on multiple rootstocks, to assess potential incompatibility and to enhance field-survival of each selection. Such plantings were established at the Ft. Pierce farm from 2000-2004, with trees of 54 diverse scion pairs planted on both mandarin (Sun Chu Sha, SCS) and trifoliate hybrid (Swingle, Carrizo, US-812, US-942, US-952) rootstocks. This site is Pineda sand, has never been limed, and is exclusively irrigated with shallow well water of pH 7.2 and 240 mg/L CaCO3 equivalents. The experimental site displayed widespread HLB that is markedly hampering tree health. HLB was identified at this site in 2006 and has affected virtually the entire grove. Tree height, width, t...

Chilling injury (CI) is a physiological disorder that is occasionally reported on fresh citrus shipments from Florida. It is most often characterized by areas of the peel that collapse and darken to form pits. Pitting is not targeted to the oil glands. Less severe symptoms may show up as circular or arched areas of discoloration or scalding. Packers and shippers should keep in mind several factors that influence if and to what degree grapefruit develop CI. This document is HS935, one of a series of the Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Publication date: July 2003. HS935/HS191: Chilling Injury of Grapefruit and its Control (ufl.edu)

Huanglongbing (HLB) and Asian citrus psyllid (ACP) are widespread at the Ft. Pierce USDA citrus grove, which is managed primarily as a site for research on HLB control and resistance. The variety collection at this grove includes rootstock cultivars grown as scions and grafted onto Sun Chu Sha, and it has become apparent that many trifoliate hybrids have much weaker symptoms of HLB than the monofoliate rootstock suckers. Therefore Poncirus trifoliata and its hybrids were tested for Candidatus Liberibacter asiaticus (CLas )16S rDNA by qPCR. In Nov. 2009 random quadrant leaf-samples and a diagnostic “most symptomatic leaf” sample were collected from the test tree canopies, along with a random sample and diagnostic sample from rootstock suckers. Data were expressed as abundance of CLas relative to citrus dehydrin. The two P. trifoliata had non-detectable or low CLas abundance, as did two citranges, except that diagnostic samples and rootstock samples from the same citranges had a very ...

The University of Florida and USDA/ARS have active citrus scion breeding programs. Hybrids that pass many selection steps and approach release are evaluated for horticultural traits and postharvest characteristics. Seven advanced selections and three commercial cultivars were harvested multiple times from research sites in Lake Alfred and Leesburg, FL. A panel of 10 members was trained to evaluate citrus fruit and reached an agreement for 10 descriptors of fresh tangerines. Fruit were washed, sanitized, peeled, and halved longitudinally so that one-half of each fruit was evaluated by the taste panel, and the other half was analyzed for quality parameters (total soluble solids and titratable acidity). Segments of each half fruit were then separated, cut in half, and placed in a fruit bowl to assure that each panelist would evaluate a sample composed of multiple fruits. Half segments (about 10) were served in 4-oz plastic cups, together with reference standards for sensory evaluation....

Candidatus Liberibacter asiaticus (CLas) has been associated with Huanglongbing, a lethal vector-borne disease affecting citrus crops worldwide. While comparative genomics has provided preliminary insights into the metabolic capabilities of this uncultured microorganism, a comprehensive functional characterization is currently lacking. Here, we reconstructed and manually curated genome-scale metabolic models for the six CLas strains A4, FL17, gxpsy, Ishi-1, psy62, and YCPsy, in addition to a model of the closest related culturable microorganism, L. crescens BT-1. Predictions about nutrient requirements and changes in growth phenotypes of CLas were confirmed using in vitro hairy root-based assays, while the L. crescens BT-1 model was validated using cultivation assays. Host-dependent metabolic phenotypes were revealed using expression data obtained from CLas-infected citrus trees and from the CLas-harboring psyllid Diaphorina citri Kuwayama. These results identified conserved and uni...

We report on the development, evaluation, and efficient delivery of antisense oligonucleotide FANA (2′-deoxy-2′-fluoro-arabinonucleotide) RNA-targeting technology into citrus trees and potato plants for management of bacterial pathogens and arthropod pests. The FANA ASO technology is a single nucleotide strand of 20–24 nt in length that incorporates 2′F- chemically modifications of nucleotides, along with a phosphorothioate backbone and modified flanking nucleotides, in their structure called “gapmers,” produced by AUM LifeTech., Inc. These unique modified structures of FANA “triggers” enables gymnotic activity that self-delivers into cells, moving systemically in treated plants and insects, with significant suppression of their RNA targets. Reported is the FANA suppression of two plant-infecting bacterium Candidatus Liberibacter asiaticus, CLas (in citrus trees), and C. Liberibacter solanacearum, CLso (in potato and tomato). The CLas pathogen is associated with huanglongbing (a.k.a...

Huanglongbing (HLB), one of the most devastating diseases of citrus is caused by the α-Proteobacteria Candidatus Liberibacter. Diplodia natalensis Pole-Evans is a fungal pathogen which has been known to cause a postharvest stem-end rot of citrus, the pathogen infects citrus fruit under the calyx, and the fruit decay typically occurs following harvest and is exacerbated by exposure to ethylene. In this study, we report that high incidence of Diplodia infection in HLB symptomatic fruit. The incidence of SER in 300 ʻHamlinʼ and 300 ʻValenciaʼ orange fruit, with or without ethylene treatment was determined. Two weeks following exposure to ethylene (10 ppm, four days), the incidence of SER in HLB-symptomatic fruit was as high as 66.7% (ʻHamlinʼ) and 58.7% (ʻValenciaʼ); whereas for asymptomatic fruit, less than 10% of the fruit were affected by SER. Confirmation of Diplodia in calyx abscission zone was by qPCR validation of the isolates and morphology of conidia.

Huanglongbing (HLB) is severely impacting Florida citrus. Productivity declines in many HLB-affected genotypes, often with greatly thinned canopies. Fruit size and quality are often adversely affected as the disease advances. HLB was assessed in diverse cultivars in commercial groves with high HLB-incidence. ‘Temple’ had the lowest HLB symptoms and Liberibacter (Las) titer, while ‘Murcott’ and ‘Minneola’ had the highest. The USDA Ft. Pierce, FL farm is managed to reveal genotype responses to HLB. Some current cultivars and hybrid seedlings demonstrate resistance/tolerance, at least to strain(s) of Las present. C. trifoliata is the best documented citrus resistance source with Las titers suppressed even when C. trifoliata is grafted onto severely-infected rootstocks. Some cultivars and hybrids have abundant foliage symptoms, but full canopies and seemingly normal fruit set and size. In 3-years of data from a replicated trial of ‘Triumph’(T), ‘Jackson’(J), ‘Flame’(F), and ‘Marsh’(M), ...

Author(s): McCollum, Greg; Hilf, Mark; Irey, Mike | Abstract: A survey of seven citrus cultivars (Citrus sinensis, C. paradisi, ‘Temple’ tangor, ‘Minneola’ and ‘Orlando’ tangelos, and ‘Fallglo’ and ‘Sunburst’ mandarin hybrids) growing in commercial citrus orchards in Florida revealed a strong correlation between HLB incidence and severity and Candidatus Liberibacter asiaticus (CLas) titer (Stover and McCollum, 2011). Temple tangor and grapefruit consistently exhibited the least severe HLB symptoms and lowest CLas titers, followed in increasing order of HLB symptoms by ‘Fallglo’ and C. sinensis.In the orchard, Candidatus Liberibacter asiaticus (CLas) is transmitted to citrus hosts via the Asian citrus psyllid (ACP); however, in most greenhouse studies CLas inoculation of citrus has been conducted by grafting. In an attempt to more closely mimic the natural process of CLas transmission by ACP to citrus we conducted a greenhouse study that included CLas-infected citron (Citrus medica) ...

Asiatic citrus canker (ACC, caused by Xanthomonas citri subsp. citri, [XccXcc]) is a serious disease of citrus, reducing yield and impacting marketability of fresh fruit. ACC has phytosanitary implications for fresh fruit trade. Lesions on fruit, leaves, and shoots in an orchard of ACC-susceptible grapefruit was monitored for production of Xcc from June to the following January in 2009 and 2010. Lesions on fruit post-harvest either packingline-treated or not treated were also monitored for production of Xcc on fruit harvested in November 2013 and January 2014. Shoot lesions generally produced fewer bacteria and had a smaller proportion of active lesions compared to leaf and fruit lesions. As the season progressed the proportion of active lesions on fruit and shoots gradually declined. Bacterial flux density (BFD) in surviving lesions was >10³ on all organs at all sample times. A window-pane analysis revealed weather variables associated with bacterial survival within the lesion. ...

Citrus crops have a long history of cultivation as grafted trees on selected rootstock cultivars, but all current rootstocks have significant limitations and traditional methods of rootstock breeding take at least 2–3 decades to develop and field test new rootstocks. Citrus production in the United States, and other parts of the world, is impaired by a wide range of biotic and abiotic problems, with especially severe damage caused by the disease huanglongbing (HLB) associated with Candidatus Liberibacter asiaticus. All major commercial citrus scion cultivars are damaged by HLB, but tree tolerance is significantly improved by some rootstocks. To overcome these challenges, the USDA citrus breeding program has implemented a multi-pronged strategy for rootstock breeding that expands the diversity of germplasm utilized in rootstock breeding, significantly increases the number of new hybrids evaluated concurrently, and greatly reduces the time from cross to potential cultivar release. We ...

Citrus tatter leaf virus (CTLV) is a graft-transmissible capillovirus that causes the tatter leaf disease of citrus and bud union incompatibility between scion and trifoliate hybrid rootstocks which can result in serious economic losses. Although several CTLV detection protocols have been described, there is a need for a highly sensitive protocol that can be used in routine diagnostic tests for virus-free budwood certification programs. To address this need, a TaqMan® chemistry based real time reverse transcription-polymerase chain reaction (RT-qPCR) assay was developed to detect CTLV from citrus plants. The nucleotide sequence analysis based on full genomic sequences of eight CTLV isolates available from GenBank showed that 3′ region of the viral genome is more conserved among different CTLV isolates than the rest of the viral genome. A new set of primers and a TaqMan® probe were designed from a conserved 500 nt-long fragment at the 3′ end of the CTLV genome for the detection of CTLV by RT-qPCR. The newly designed primers for CTLV RT-qPCR assay had ca. 98.6% amplification efficiency and showed 100% CTLV detection rate when they were tested for previously known CTLV-positive trees infected with different CTLV isolates. The sequencing of 132 bp-long RT-qPCR amplicon followed by BLASTn search showed that it had 94% to 98% sequence identity to those of various CTLV isolates available in GenBank, confirming the specificity of the assay to reliably detect CTLV.

Plant microbiomes are critical components to plant health and can influence disease outcomes. We provide empirical data describing disease-induced shifts within the citrus microbiome at different levels of huanglongbing (HLB) disease severity. HLB is associated with an invasive phloem-limited bacterium, ‘Candidatus Liberibacter asiaticus’, that is introduced into the aerial portions of the tree by an insect vector. Disease manifests as aboveground foliar and fruit symptoms and significant root decline belowground. During the early phase of disease, there were depletions of putative keystone taxa in leaves and roots, followed by enrichments of putative beneficial taxa, suggesting a microbially derived immune response involved in plant protection that is ancillary to immune components encoded in the plant’s genome. In the late phase of disease, we observed enrichments of parasitic and saprophytic microorganisms, particularly in the roots. The community shifts within the root compartme...

The fungicides thiabendazole (TBZ) or imazalil were applied at 1 g·liter-1 at 24 or 53C to `Marsh' and `Redblush' grapefruit (Citrus paradisi Macf.) to reduce fruit susceptibility to chilling injury (CI) and decay. Generally, there was more CI and decay on `Marsh' grapefruit than on `Redblush'. Severity of CI was lower in grapefruit that had been dipped at 53C than at 24C. Fruit dipped in fungicides had less CI than fruit dipped in water alone. Imazalil was more effective in reducing CI than TBZ. Fungicides reduced decay at both temperatures, and imazalil was better than TBZ. Chemical names used: 2-(4-thiazolyl)benzimidazole (thiabendazole, TBZ); 1-[2-(2,4-dichlorophenyl)-2-(2-propenyloxy)ethyl] -1H -imidazole (imazalil).

Mature, green tomatoes were either gassed or not gassed with C2H4 for 24 h, immersed in 42C water for 60 min, or held in 38C air for 48 h or not treated, and then stored at either 2C or 13C for 14 days before ripening at 20C. During ripening, the fruit were evaluated for color development, internal quality, and decay and for volatiles when full ripe. Both high-temperature treatments reduced chilling injury and inhibited decay. Days to ripen after removal from storage at 2C or 13C was not influenced by heat treatment method. Color development, lycopene content, and internal quality characteristics of fruit were similar at the ripe stage, irrespective of heat treatment. Of 15 volatiles analyzed, seven showed decreased levels of concentrations as a result of C2H4 gassing, nine showed decreased levels when stored at 2C prior to ripening, and most were unaffected by the heat treatments. Heat treatments appear to be beneficial for maintaining tomato fruit quality.

The influence of postharvest heat conditioning at 38 °C for 24, 48, or 72 hours on ripe `Gialla' cactus pear [Opuntia ficus-indica (L.) Miller] fruit produced by the spring flush was investigated during 21 days of storage at 6 °C and 90%-95% relative humidity (RH) followed by 7 days at 20 °C and 70%-75% RH (simulated marketing). Conditioning for 24 to 72 h reduced by 50% the severity of chilling injury (CI) on cactus pears following exposure to cold storage. Treatment for 24 to 72 h was also effective in reducing decay, with conditioning for 24 h being the most effective. Overall visual quality was better in heat-conditioned compared with control fruit. Mass loss was significantly reduced by all heat conditioning treatments. Respiration rate was not affected by heat treatment. Ethylene evolution was lower in fruit heat-conditioned for 48 or 72 h than for 0 h. Conditioning for 72 h resulted in the highest fruit ethanol levels. The influence of conditioning on juice pH, titratable...

We determined the effects of 1-methylcyclopropene (1-MCP) and ethylene on color change and CO2 and ethylene production in grapefruit. Treatment with 1-MCP at concentrations equal to or greater than 75 nL·L−1 inhibited ethylene-induced degreening, but increasing 1-MCP concentrations greater than 150 nL·L−1 did not cause additional inhibition of degreening. Although ethylene-induced degreening was inhibited by 1-MCP, the effect was transient. Treating grapefruit with 15 to 75 nL·L−1 1-MCP resulted in a slight suppression of CO2 production, whereas treatment with 150 or 300 nL·L−1 1-MCP resulted in rates of CO2 production significantly higher than nontreated fruit. 1-MCP treatment also caused a very pronounced increase in the rate of C2H4 production that was both dose- and time-dependent. The effects of 1-MCP on respiration and ethylene evolution were reduced if fruit was subsequently exposed to ethylene. Fruit treated with 1-MCP alone had the highest rates of CO2 production, fruit tre...