Research article
Metabolically speaking: Possible reasons behind the tolerance of ‘Sugar Belle’ mandarin hybrid to huanglongbing

https://doi.org/10.1016/j.plaphy.2017.05.001Get rights and content

Highlights

  • Field observations showed that a new mandarin hybrid, ‘Sugar Belle’ mandarin is tolerant to the HLB disease.

  • We studied the volatile and non-volatile metabolites of greenhouse-reared ‘Sugar Belle’ mandarin and four of its ancestors.

  • Sugar Belle was high in thevolatiles, α-thujene, para-cymene, γ-terpinene, thymol, β-elemene, and t-β-caryophyllene.

  • Sugar Belle was high in synephrine, phenolic compounds, chiro-inositol, fructose, glucose, and sugar acids.

  • The high levels of antimicrobial volatiles phenolic compounds might be the reason behind the tolerance to HLB.

Abstract

Huanglongbing (HLB) is currently considered the most destructive disease of citrus. Since its spread to the Americas, HLB has killed millions of trees and caused a sharp decline in production in many citrus growing regions. With the continuous spread of HLB disease in Florida and worldwide, there is an urgent need for the development of commercial citrus cultivars with a strong tolerance to HLB. Interestingly, field observations showed that some of the recently released mandarin hybrids such as ‘Sugar Belle’ were tolerant to HLB. In this study, we investigated the volatile and non-volatile metabolites of greenhouse-grown ‘Sugar Belle’ mandarin and four of its ancestors in order to understand why ‘Sugar Belle’ mandarin is relatively tolerant to HLB. Leaf volatiles were directly extracted with hexane and analyzed using gas chromatography-mass spectrometry (GC-MS). Leaf polar metabolites were extracted with a mixture of methanol:water (1:1, v/v), derivatized to their trimethylsilyl ethers, and analyzed using GC-MS. Forty-seven volatile compounds and forty-two polar metabolites were detected in ‘Sugar Belle’ mandarin leaves and its ancestors. ‘Sugar Belle’ was high in several volatiles such as α-thujene, para-cymene, γ-terpinene, thymol, β-elemene, and (E)-β-caryophyllene. Some of these volatiles, especially thymol, β-elemene, and (E)-β-caryophyllene are known for their anti-microbial activity. In addition, ‘Sugar Belle’ mandarin was the highest in synephrine, benzoic acid, ferulic acid, caffeic acid, chiro-inositol, fructose, glucose, threonic acid, saccharic acid, and galactaric acid, and the second in threonine, malic acid, and myo-inositol compared to the ancestors. Phenolic compounds such as benzoic, ferulic, and caffeic acids may act as antibacterial agents, whereas others like sugar alcohols may protect ‘Sugar Belle’ mandarin from stress during pathogen attack. The tolerance of ‘Sugar Belle’ and other newly released mandarin hybrids should be further evaluated using greenhouse controlled studies. If tolerance of these hybrids is confirmed, they could be used to replace the traditionally susceptible cultivars.

Introduction

Huanglongbing (HLB), also called citrus greening, was first discovered in southern China in 1919 (Bové, 2006). North America was free of HLB until it was reported in Florida, USA in 2004 (Bové, 2006). Since its discovery in Americas, HLB has spread in many citrus growing regions in Brazil and U.S.A (Teixeira et al., 2008). HLB has been responsible for the death of millions of citrus trees and it resulted in a sharp decline in citrus production worldwide (Hodges et al., 2014). Due to the spread of HLB disease in Florida, Florida's orange production during 2015–2016 season was the lowest in the past fifty years (NASS, 2017).

Candidatus Liberibacter, the actual agent for citrus greening, is a phloem-limited, Gram-negative bacterium. Three species of yet-uncultured Candidatus Liberibacter were associated with citrus greening: Candidatus Liberibacter asiaticus (CLas), Candidatus Liberibacter americanus (Clam), and Candidatus Liberibacter africanus (CLaf). The CLas has been found in Asia, North America, and Brazil, while Clam and CLaf are endemic to Brazil and Africa, respectively. CLas and Clam are transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama, whereas CLaf is transmitted by the African citrus psyllid, Trioza erytreae (Tatineni et al., 2008).

Although Candidatus Liberibacter can be spread by grafting, psyllids are the main transmission vector of the CLas pathogen in the field. Psyllids acquire the citrus greening pathogen while feeding on citrus phloem sap of the infected trees then transmit it by moving to healthy trees. Field observation showed that D. citri prefers some host plants more than others, for example Poncirus trifoliata was an occasional host, while sweet orange and Murraya paniculata (L.) Jack (orange jasmine) were the most preferred hosts for D. citri (Halbert and Manjunath, 2004). In addition, comparative laboratory studies showed that some host plants were more preferred by D. citri than others. Tsai and Liu tested the preference of D. citri to several host plants and found that grapefruit was the most preferable host, followed by rough lemon, orange jasmine, and sour orange (Tsai and Liu, 2000). Richardson and Hall also showed that hybrids of Poncirus trifoliata x Citroncirus spp. were more tolerant to D. citri than Citrus macrophylla Wester (Alemow). Although, they did not investigate the mechanism behind the tolerance of P. trifoliata to D. citri, it is believed that P. trifoliata may have antixenotic resistance to D. citri (Richardson and Hall, 2014).

Although there are no commercial citrus cultivars with strong tolerance to CLas, which makes it remarkably difficult to apply effective control measures (Fan et al., 2013), field observations showed that some citrus cultivars are more tolerant to CLas bacteria than others (Halbert and Manjunath, 2004). In addition, greenhouse controlled studies also showed that some citrus cultivars are more tolerant to CLas pathogen (Folimonova et al., 2009). Sensitive cultivars showed severe leaf chlorosis, decreased growth and death, while the tolerant cultivars showed fewer symptoms (Folimonova et al., 2009). In another study, it was also found that some Malaysian citrus cultivars were also more tolerant to CLas pathogens than others (Shokrollah et al., 2009).

In our previous work, we investigated the phloem sap composition of fourteen different citrus cultivars with different degrees of tolerance to CLas pathogen. We reported that the levels of most of the amino acids, especially those implicated in plant defense to pathogen attack such as phenylalanine, tryptophan, and tyrosine were higher in CLas-tolerant cultivars (Killiny and Hijaz, 2016). In another study, we investigated the volatile composition of the same citrus cultivars and indicated that citrus leaf volatiles could play a role in citrus tolerance against the CLas pathogen. Tolerant cultivars contained higher amounts of volatiles especially those with strong antimicrobial activities such as aldehydes than susceptible cultivars (Hijaz et al., 2016).

To date, there is no effective tool to stop the spread of the CLas pathogen or manage HLB disease. Management mainly depends on the control of D. citri vector with insecticides, geographical isolation, use of pathogen-free nursery trees, and removal of infected trees to reduce inoculum (Gottwald et al., 2012, Halbert and Manjunath, 2004). Although removal of infected plants was proposed for eradication of HLB, this strategy is problematic and expensive (Halbert and Manjunath, 2004). Since the previous management strategies were unable to stop the fast spread of HLB, commercial citrus producers have tried various means to mitigate the disease (Gottwald et al., 2012). Recently, enhanced nutritional programs (ENPs) were proposed to minimize the deleterious effects of HLB on infected trees and maintain their productivity. However, well-designed field studies showed that the ENP was not able to maintain long-term tree health, productivity, or fruit quality (Gottwald et al., 2012). On the other hand, it is believed that the use of ENP strategies may increase the spread of the CLas pathogen since it prolongs tree life but does not affect the CLas titer (Gottwald et al., 2012).

Field observations showed that some of the recently released mandarin hybrid cultivars were tolerant to the HLB disease (Stover et al., 2016). If the tolerance of these mandarin hybrids is confirmed, they could be used to replace the traditional susceptible cultivars such as sweet orange and grapefruit (Stover et al., 2016). In 2009, the University of Florida IFAS/CREC cultivar improvement team released many mandarin hybrids including the ‘Sugar Belle’ previously known as LB8-9 [‘Clementine’ mandarin (Citrus reticulata) x ‘Minneola’ tangelo (Citrus x Tangelo), ‘Duncan’ grapefruit (Citrus paradisi) x ‘Dancy’ tangerine (Citrus reticulata)]. ‘Sugar Belle’ mandarin is a mid-season mandarin that matures between late November and early January under Florida conditions. An earlier evaluation of ’Sugar Belle’ mandarin cultivar and ‘Sunburst’ tangerine and ‘Minneola’ tangelo revealed that ‘Sugar Belle’ mandarin decay was lower compared to ‘Sunburst’ tangerine and its peel, juice color, and vitamin C content were higher than ‘Minneola’ tangelo (Dou and Gmitter, 2007).

Recently published data from Stover et al. (2016) on the field tolerance of several cultivars to HLB, suggested that growth and yield were not suppressed in ‘Sugar Belle’ mandarin trees even in the presence of HLB symptoms, which indicated that ‘Sugar Belle’ mandarin could be tolerant to the HLB disease. In addition, field observation indicated that ‘Sugar Belle’ mandarin is tolerant to HLB regardless of the rootstock (Dr. Tripti Vashisth, CREC-IFAS-UF, personal communication). Surprisingly, field observation showed that ‘Sugar Belle’ mandarin was a good host for D. citri (observation of Dr. Fred G. Gmitter, Jr.), suggesting that ‘Sugar Belle’ mandarin may have an innate antimicrobial resistance.

With the current epidemic spread of HLB in the United States as well as in many citrus producing countries, there is an urgent need to provide growers with commercial citrus cultivars that are tolerant to HLB. Our objective was to evaluate the volatile and non-volatile metabolites of ‘Sugar Belle’ mandarin which shows tolerance to HLB (Stover et al., 2016) and its parents which were evaluated as susceptible cultivars (Folimonova et al., 2009, McCollum et al., 2016) in order to identify which compounds may be involved in tolerance to CLas. Correlating plant metabolites to HLB tolerance, by identifying specific compounds with anti-microbial activity in naturally tolerant cultivars, may provide important information about plant−pathogen interactions. In addition, results from these studies may open new avenues to improving the disease tolerance/resistance for the highly susceptible cultivars via biotechnology applications.

Section snippets

Plant material

The budwood for ‘Sugar Belle’ mandarin, previously known as LB8-9 and its parents, [‘(‘Clementine’ mandarin (Citrus reticulata) × ‘Minneola’ tangelo, ‘Duncan’ grapefruit (C. paradisi) × ‘Dancy’ tangerine (C. reticulata))] (Fig. 1) were obtained from Chiefland Budwood Foundation at Chiefland, Florida. The degree of tolerance of selected cultivars to CLas pathogen is shown in Table 1. The budwoods were side-grafted onto one-year-old UF-15 rootstocks [Hirado Buntan pummelo (Citrus maxima) x

Leaf volatiles

Forty-seven volatile compounds were detected in the hexane extracts of ‘Sugar Belle’ mandarin leaves and its ancestors (Table 2). Linalool, γ-terpinene, α-pinene, β-elemen, Z-β-ocimene, β-pinene, and thymol were the major volatiles in ‘Sugar Belle’ mandarin and they made up more than 84% of the total volatile compounds (Table 2). Linalool, sabinene, γ-carene, β-pinene, d-limonene, geraniol, and geranial were the most abundant volatiles in ‘Clementine’ mandarin and they made up more than

Discussion

Although field and greenhouse studies showed that some citrus cultivars are more tolerant to CLas pathogen than others, the mechanism underpinning this tolerance has not been determined yet (Folimonova et al., 2009). Understanding of the mechanisms behind citrus tolerance to CLas could help in developing commercially tolerant citrus cultivars. In this study, we investigated the leaf volatile and non-volatile metabolites of ‘Sugar Belle’ mandarin and its ancestors in order to identify

Contribution

Nabil Killiny conceived the study. All authors contributed in conducted the experiments and analyzing of data. Writing of the manuscript was by all authors. Nabil Killiny obtained the funding for the work.

Acknowledgments

We thank our lab members for the helpful discussion and technical assistance. This work was generously funded by a grant #126409-G003523 from NIFA-SCRI for NK.

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