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Current Knowledge on Huanglongbing (HLB) and the Interactions of the Pathogen, Vector, and Host

Research on citrus huanglongbing (HLB) and its causal agents, vectors, and hosts has been going on for many decades, and while controlling HLB remains a major challenge, much has been learned about this disease. Although not a comprehensive review of what is known about HLB since its first reported occurrence in the early 1900s, this chapter provides key information about the disease that reflects the current understanding of the factors that influence its occurrence, severity, and incalcitrance to effective management.

HLB: THE CAUSAL (OR ASSOCIATED) BACTERIA

Candidatus Liberibacter asiaticus

Candidatus Liberibacter asiaticus (CLas), the gram-negative, walled bacterium to which citrus greening (HLB) in the United States is attributed, is a member of the family Rhizobiaceae¹ in the phylum Proteobacteria. Two closely related bacteria also believed to cause HLB, Ca. Liberibacter africanus (CLaf) and Ca. Liberibacter americanus (CLam), have been characterized from other geographical locations and named for the continent on which they were first found. All three species are nonculturable and phloem restricted in planta, leading to the “Candidatus” taxonomic status.

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¹ A family of Proteobacteria having a range of impacts on plants, some fixing nitrogen after becoming established inside root nodules of legumes, others causing plant disease by genetically engineering the plant host.

Genetic and Morphological Characteristics

Understanding the plant-colonizing and disease-inducing mechanisms of CLas has been hampered by the inability to grow it in the laboratory, and much of what is known or hypothesized has been deduced from multiple genome sequences of the three HLB-associated bacteria. A critical feature for both survival and pathogenicity of the bacteria is the ability to secrete cytoplasmically synthesized enzymes and virulence factors through the cell plasmalemma (a plasma membrane that bounds a cell, especially one immediately within the wall of a plant cell).

Most of the known CLas strains harbor two nearly identical prophages, SC1 and SC2 (Zhang et al., 2011). SC1 replicates, produces phage particles, and becomes lytic in the plant host (but not in the psyllid vector), while SC2 lacks lysis-associated genes and exists as a replicative excision plasmid (a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently). The CLas phage SC2 encodes two putative peroxidases believed to contribute to host defense responses by scavenging reactive oxygen species (Zhang et al., 2011) and downregulating genes involved in H₂O₂-mediated defense signaling in plants (Jain et al., 2015), thereby suppressing host symptom development.

The genome of CLas, at 1.23–1.15 Mb (Duan et al., 2009; Zheng et al., 2014), is significantly smaller in size and has fewer genes and lower guanine and cytosine content than related culturable bacteria. Sequence annotation indicated that these features are reflective of the loss of a number of functions during the microbe’s adaptation to its intracellular parasitic lifestyle within the host plant and insect vector. Several genes related to DNA and excision repair are lacking, and the species has only a small subset of sigma factors (bacterial transcription initiation-related proteins that enable specific binding of RNA polymerase to gene promoters) involved in transcription (Hartung et al., 2011). Some metabolic enzymes, including those required for purine and pyrimidine² metabolism, also have been lost (Hartung et al., 2011).

Disease Development and Symptoms

Because noncultivability of CLas and its HLB-associated relatives prevents the completion of Koch’s postulates, the question remains whether these bacteria are solely responsible for HLB or whether they are components of a disease complex (Sechler et al., 2009). In fact, phytoplasmas have also been associated with the disease in Brazil (Teixeira et al., 2008)

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² A purine is a heterocyclic aromatic organic compound consisting of a pyrimidine ring and an imidazole ring, whereas a pyrimidine is an aromatic heterocyclic organic compound containing two nitrogen atoms; both are components of nucleic acids.

and China (Chen et al., 2009), frequently in mixed infections with Ca. Liberibacter spp. What role, if any, other microbes play in the disease remains to be clarified.

Initial tree symptoms are frequently the appearance of yellow shoots and blotchy, mottled leaves, sometimes with vein yellowing. Symptoms may differ on opposite halves of a leaf. As the bacteria translocate and colonize the plant systemically the canopy progressively becomes chlorotic, tree growth slows, leaves remain smaller than normal, and leaf tips become necrotic (Timmer et al., 2000; da Graça and Korsten, 2004; Halbert and Manjunath, 2004; Bové, 2006; Gottwald et al., 2007). Often, parts of the tree remain healthy or symptomless such that the disease has a sectored appearance. Leaves can thicken and veins enlarge and appear corky. Later, yellow blotches appear between veins that remain green, similar to zinc deficiency,³ and leaves may drop as twig ends become necrotic (Gottwald et al., 2007).

Fruit from diseased trees fails to achieve full size, may be asymmetrical and have areas on the surface that remain green, and is marked by bitter taste. Seeds may be aborted. As the disease progresses fruit yield and quality decline (Timmer et al., 2000; Halbert and Manjunath, 2004; Bové, 2006), eventually falling below an economically tolerable level. All species of the family Rutaceae are potential hosts. Historically, the most susceptible are sweet oranges (Citrus sinensis L. Osb.), tangelos (Citrus × tangelo), and mandarins (C. reticulata Blanco). Moderately susceptible hosts have included grapefruits (C. paradisi Macf.), lemons (Citrus × limon), Rangpur lime (Citrus × limonia Osb.), calamondins (X Citrofortunella microcarpa), and pomelos (C. maxima). Mexican limes or key limes (Citrus × aurantifolia Swingle) and trifoliate orange (C. trifoliata or Poncirus trifoliata) may be even more tolerant. Noncitrus Rutaceae species, such as Murraya paniculata, also serve as hosts of the HLB-associated pathogens (Timmer et al., 2000; Appel, 2004; da Graça and Korsten, 2004; Halbert and Manjunath, 2004; Lopes et al., 2005; Bové, 2006). The symptoms of HLB caused by the American (CLam) and African (CLaf) bacteria are very similar to those caused by CLas, but environmental optima differ, CLaf developing under cool temperatures (20ºC–25ºC) and CLam under a wider temperature range (20ºC–35ºC).

After inoculation into the citrus phloem sieve tubes by the psyllid vector, CLas population levels increase rapidly, peaking at approximately 200 days postinoculation and reaching about 10⁸ cells per gram of plant tissue (estimated by copies of 16S ribosomal DNA) (Coletta-Filho et al., 2014). However, infected trees remain asymptomatic through the early disease

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³ Zinc deficiency symptoms include reduced leaf size, narrow leaves, yellow mottled on green background, and decreased overall fruit yield.

stages, and early symptoms are difficult to recognize because of their mildness and resemblance to other conditions.

HLB: THE PSYLLID VECTOR

Asian Citrus Psyllid

Geographic Distribution and Invasion

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), originally from India or other parts of Asia,⁴ has been recognized as an invader in North America and Latin America for several decades. In 1998, it was initially discovered in the Caribbean Basin, on the island of Guadeloupe (Étienne et al., 1998), and in South Florida (Halbert, 1998; Halbert et al., 2002). Since then, populations have spread widely among islands and other countries in the Caribbean Basin, including the Bahamas, the Caymans, Cuba, Dominican Republic, Jamaica, Mexico, Puerto Rico, and Venezuela (Halbert and Nuñez, 2004). In the continental United States its range now extends from South Carolina to Florida in the south; and from Georgia westward to Alabama, Arizona, Louisiana, Mississippi, southern California, and Texas (USDA APHIS, 2017). It has also been found in the Hawaiian Islands, on Hawaii, Kanai, Lanai, Maui, Molokai, and Oahu (Conant et al., 2009). In the insect’s native range (e.g., Asia), ACP infests native citrus but rarely if ever causes direct damage as a result of feeding. Importantly, the insect affects citrus as a result of its capacity to persistently transmit different species of HLB-associated bacterial pathogens classified as Ca. Liberibacter spp. (Yang et al., 2006). Two separate ACP introductions (founding events) have likely occurred in the Americas, one each in South and North America, whereas North America and Hawaii appear to share a similar source of invasion. In most of the areas where the ACP has become established, it has spread rapidly in residential and commercial plantings through both natural and human-assisted transport. Across nearly all of Florida, populations of ACP are well established, and now active populations have been detected in many areas of California. Since the initial introductions, recent phylogeographic⁵ investigations of the ACP mitochondrial cytochrome oxidase subunit I gene have revealed variations in psyllid populations collected in the Americas and the Pacific region (Boykin et al.,

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⁴ The origin of ACP is discussed at length by Beattie et al. (2008) in their paper “On the Origins of Citrus, Huanglongbing, Diaphorina citri and Trioza erytreae” available at https://www.plantmanagementnetwork.org/proceedings/irchlb/2008/presentations/IRCHLB.K.2.pdf. Accessed May 4, 2018.

⁵ Pertaining to the study of the historical processes responsible for geographic distributions of individuals by considering population genetics.

2012). Specifically, 23 ACP haplotypes⁶ were identified, and these fell into two groups: from South America (group 1) and from North America and Hawaii (group 2).

Psyllid Biology: Description, Communication, Life Cycle, and Reproduction

Psyllids, or jumping plant lice, have been classified historically as members of the family Psyllidae (a family composed of homopterous insects), although recently the group has been recognized as a superfamily comprising eight families, including the Liviidae, to which D. citri belongs (Burckhardt and Ouvrard, 2012). Similar in size to aphids, psyllids are typically more host specific. They commonly infest new flush growth or tender leaves. Consistent with their common name, the jumping plant lice are generally more active than aphids, readily propelling themselves into the air by rapid movements of their hind legs (Burrows, 2012) or by taking flight if disturbed. Psyllid antennae usually possess 10 segments and lack abdominal cornicles, which are unique to the more sedentary aphids. Adult ACPs are approximately 3.5 mm in length and have mottled brown bodies. Forewings of the adults are broader distally and mottled, with a brown band that encircles the outer half. Antennae have black tips with two small, light brown spots on the middle segment of the flagella. Older adults are covered with a whitish waxy secretion, giving the adult insect a dusty appearance (Martin et al., 2012). Adult ACPs are oviparous and eggs are very small, elongate, and thicker at the base and tapered toward the distal end (Martin et al., 2012).

Like other psyllid species, the ACP depends on multimodal signaling for mate finding and courtship (Stockton et al., 2017a). Depending on plant species, volatiles from intact or psyllid-damaged foliage can act as long-distance attractants for male and/or female adults searching for host trees and populations of conspecifics (Gharaei et al., 2014). Males rapidly vibrate their wings to send substrate-borne signals to females, who respond in kind (Wenninger et al., 2008) in a behavior called duetting (Mankin et al., 2013). After this exchange of vibrational signals, cuticular hydrocarbons produced by females appear to function as short-range orientation cues for males. Which cuticular constituents are responsible for male attraction remains unclear, but dodecanoic acid, which is present in cuticle extracts, was attractive to males in laboratory bioassays. Moreover, traps baited with dodecanoic acid in the field attracted more males at the highest concentration tested than did traps without the compound (Mann et al., 2012a).

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⁶ A haplotype (haploid genotype) is a particular combination of markers (alleles) at multiple locations on a chromosome, used in assessing relatedness among individuals.

Experience can modify behavioral responses to chemical signals; prior to sexual maturity, which typically is achieved 3 to 4 days posteclosion (emergence from the pupal case or hatching from the egg), both males and females appear to engage in “practice courtship and copulation behaviors” (Stockton et al., 2017a); as well, after successfully mating, males are more responsive to female odors. Females are capable of mating with multiple males, but the presence of multiple males over a period of weeks may reduce oviposition success (Wenninger et al., 2008). During mating males can transmit CLas to uninfected females (Mann et al., 2012a).

Female ACPs require multiple matings throughout their adult life to maintain viable eggs and begin to lay eggs as soon as 1 day after mating. Eggs are laid on the emergent tips of new citrus shoots (e.g., “feather flush” growth) on and between unfurling leaves, and adult females may lay more than 800 eggs during their lives (Liu and Tsai, 2000). Newly laid eggs are oriented with the long axis vertical to the leaf surface; they are initially pale in color, later turning yellow and finally orange before hatching. Eggs hatch within 48 to 96 hours. Nymphs are significantly smaller than adults, and the five nymphal instar stages are typically completed in 11 to 15 days. Instars are initially light yellow in early stages, transitioning to yellowish orange in later stages. There are no abdominal spots but the wing pads are large, and filaments are confined to the apical plate of the abdomen. The total life cycle requires between 15 and 47 days, depending upon environmental conditions, and newly emerged adults can live for several months (Grafton-Cardwell et al., 2013).

Dispersal and Seasonal Phenology

There is no known ACP diapause, but activity of adult populations, as measured by sticky cards and beat samples, is considered low in winter months and prior to spring flush in citrus. Estimated through field-based life history investigations, the ACP has 9 to 10 generations per year, whereas in field cage evaluations up to 16 generations have been recorded (Hall et al., 2008; Hall and Hentz, 2010). The nymphs, which are nearly always found on new growth, move in a slow, steady manner when disturbed. The adults can jump or leap greater distances when disturbed and may even fly short distances. Adults are usually found in aggregations on lower leaf surfaces with their heads often pressed against the abaxial (lower) surface of new leaves. The time of year of greatest psyllid activity corresponds with the periods of new flush growth in citrus, and some variation in this activity is concomitantly associated with variation in citrus varietal flush patterns (Hall and Hentz, 2010; Khan et al., 2014). Investigations of disease progress in citrus have made inferences about the timing and distance of ACP dispersal that suggest that adult flights can regularly occur over 25- to 50-m

distances. In portions of Southeast Asia, dispersal of nearly 500 km has been suggested, resulting from major synoptic weather patterns (Khan et al., 2014; Lewis-Rosenblum et al., 2015). Protein marking has revealed that adults can regularly move distances approaching 100 m in 72 hours and that movement into managed citrus groves in Florida often resulted from invasion from adjacent abandoned citrus in Florida (Kobori et al., 2011; Hall and Hentz, 2014).

Once dispersing adults have located suitable host plants for feeding or oviposition, trivial plant-to-plant movements appear to constitute most of their activity. Based upon preliminary trapping data, no obvious seasonal movement patterns have been described except for elevated activity around the spring flush of citrus foliage. Both managed and abandoned citrus plots are infested by adult ACPs, but flight patterns of these insects are not obviously related to either discrete edaphic (of, produced by, or influenced by the soil) or abiotic sets of conditions (e.g., wind, short-wave radiation, temperature, and humidity) (Patt and Sétamou, 2010; Hall and Hentz, 2011). Laboratory-based flight mill tests suggest that adult ACPs are able to sustain continuous flight for estimated distances of nearly 1.2 km (Arakawa and Miyamoto, 2007). The majority of adult ACPs, however, have far shorter flight times and their dispersal consists of short-duration, tree-to-tree flights. Although temporal patterns of these short-duration flights are consistent in local citrus-producing regions, no flight phenology predictions have been generated to describe patterns of movement, migration, or capture over larger landscapes. However, the elevated activity in captures may be due to seasonal variation in the timing of spring flushing in citrus, which can also vary regionally. The lack of consistency in adult ACP movement patterns underlies the need for vigilance in monitoring and surveillance of these mobile pests.

Microbial Associates and Interactions with the Asian Citrus Psyllid

The microbiome of ACP has recently been investigated by analysis of adults, nymphs, eggs, and cell cultures. Kolora et al. (2015) found evidence of 10 distinct types of bacteria, and 4 species of bacteria were detected in cell cultures with 2 new types associated with psyllids. Kruse et al. (2017) reported that ACP has three well-described bacterial endosymbionts, Candidatus Profftella armatura, Candidatus Carsonella ruddii, and Wolbachia spp., as well as other internal, extracellular bacteria. Bacterial symbionts of sap-sucking insects generally function to compensate for phloem that is nutritionally poor, specifically providing amino acids that cannot be synthesized by the host. While the prevalence of each endosymbiont in the ACP is positively correlated, their localization and function within the insect vary. Both Profftella and Carsonella are localized in collections of cells called the

bacteriome, while Wolbachia is found in somatic and reproductive tissues in addition to the bacteriome (Dobson et al., 1999; Hosokawa et al., 2010).

HLB: THE RUTACEOUS HOSTS

Citrus Origin and Taxonomy

Citrus species are widely believed to be native to subtropical and tropical regions of Asia and the Malay Archipelago.⁷ Citrus spread from these areas of origin to regions across the globe having tropical and subtropical climates (Webber, 1967). Ancient Roman literature describes citrus culture, and people of the Mediterranean region have continued to cultivate citrus trees, particularly mandarins, sweet orange, and lemons, producing fruits for local consumption and for export (Webber, 1967). Spanish conquistadors brought citrus to Florida sometime between 1513, when Ponce de León first landed in Florida, and 1565, when the city of St. Augustine was established (Webber, 1967).

The genus Citrus belongs to the family Rutaceae, which contains about 160 genera and 1,800 species. Around 7 million years ago, citrus progenitors diverged into two main genera, Citrus and Poncirus (Webber, 1967; Pfeil and Crisp, 2008). Phylogenetic and gene-sequencing studies (Scora, 1975; Barrett and Rhodes, 1976; Wu et al., 2014) indicate that most cultivated citrus, sweet oranges, grapefruit, lemons, limes (Citrus × aurantiifolia), and tangerines (C. reticulata), resulted from hybridization that occurred over several thousand years between three citrus species—Citrus reticulata (mandarins), C. maxima (pomelos), and C. medica (citrons)—and possibly a fourth species as yet unidentified. Poncirus trifoliata is native to northern China and Korea, and, while cross-compatible with citrus species, P. trifoliata differs from the latter in having deciduous compound leaves and pubescent fruit. It is significantly more cold hardy than citrus species and can be cultivated in U.S. Department of Agriculture (USDA) Plant Hardiness Zone 6.

Citrus Biology and Propagation

Citrus trees are broad-leafed evergreens adapted to sunny, humid environments having fertile soil and adequate rainfall or irrigation. Trees flower in the spring, and fruit begin to ripen in the fall or early winter months, depending on the cultivar. Citrus fruits are good sources of vitamin C

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⁷ The possible origin of citrus is explored by Beattie et al. (2008) in the paper “On the Origins of Citrus, Huanglongbing, Diaphorina citri and Trioza erytreae” available at https://www.plantmanagementnetwork.org/proceedings/irchlb/2008/presentations/IRCHLB.K.2.pdf. Accessed May 4, 2018.

and flavonoids. The vitamin C content depends on the species, variety, and mode of cultivation (Duarte et al., 2010). The flavonoids include various flavanones and, in lower concentrations, flavones and flavonols (Benavente-Garcia et al., 1997). Citrus fruits are nonclimacteric; i.e., they ripen without ethylene or respiration bursts. Respiration slowly declines during fruit development and ethylene release by the mature fruit is extremely low (Aharoni, 1968; Eaks, 1970; Goldschmidt et al., 1993). Fruits of early varieties of citrus can meet legal maturity standards before the peel attains the characteristic varietal color and therefore require degreening to enhance coloration. This is accomplished by exposure of fruit to ethylene gas, which destroys chlorophyll and allows the yellow or orange rind pigments to predominate (Ritenour et al., 2015).

Citrus species are generally not cold hardy. Mandarins (C. reticulata) tend to be the hardiest of the common Citrus species and can withstand short periods as cold as −10°C (14°F), but commercial production requires that temperatures do not fall below −2°C (28°F). Citrus fruits are less tolerant to freezing conditions than citrus trees or foliage. Freeze damage to fruit is influenced by variety, minimum temperature, and duration of temperatures (Oswalt and Hurner, 2012). Freeze injury in fruit is characterized by the appearance of water-soaked areas on the segment membranes with the juice sacs or vesicles in injured areas subsequently becoming dry and collapsed. Fruit damaged by freeze may drop quickly or over time. Freeze injury in citrus leaves, caused by ice formation in the intercellular spaces, is indicated by the appearance of water-soaked areas on the leaf surface. Ice formation in wood may result in bark splits, which, if extensive, may cause limbs to die and later break off many years after the freezing event (Zekri et al., 2016).

Citrus cultivars are propagated vegetatively by grafting to maintain the integrity of the desired cultivar. Rootstocks are selected for disease and nematode resistance, soil adaptation, resistance to flooding and drought, cold hardiness, and effects on tree size (Castle et al., 2016). Rootstocks may be seed propagated. For example, rough lemon, sour orange, P. trifoliata, and hybrids are polyembryonic and therefore can be seed propagated true to type (Castle et al., 2016).

Citrus Diseases and Insect Pests

In addition to HLB and ACP, there are a number of other diseases and pests that affect citrus. Other diseases that occur in citrus cultivated in Florida include citrus canker, which is a leaf, fruit, and stem blemishing disease caused by the bacterium Xanthomonas citri subsp. citri (Dewdney and Graham, 2017); fungal diseases that include Phytophthora foot and root rots, and brown rot of fruit, which can be caused by Phytophthora

nicotianae or P. palmivora (Graham and Dewdney, 2017; Graham et al., 2017), citrus black spot, caused by Guignardia citricarpa (Dewdney et al., 2017), Alternaria brown spot, caused by Alternaria alternata (Dewdney, 2017), and postbloom fruit drop, caused by Colletotrichum acutatum (Peres and Dewdney, 2017); viroid⁸ diseases that include exocortis and cachexia (Roberts and Brlansky, 2017); Citrus tristeza virus (CTV), which is a major cause of the decline and eventual death of trees on sour orange rootstocks (Roberts et al., 2017); and citrus blight, a disease of unknown etiology that causes trees to decline and become unproductive and affects all major rootstocks and seedlings to varying degrees (Futch et al., 2017).

Insects that affect citrus in Florida include the citrus leafminer (Phyllocnistis citrella) (Hunsberger et al., 2006); several scale insects such as the snow scale (Unaspis citri), the Florida red scale (Chrysomphalus aonidium), and the purple scale (Lepidosaphes beckii); several whitefly species such as the citrus whitefly (Dialeurodes citri), the cloudy-winged whitefly (D. citrifollii), the woolly whitefly (Aleurothrixus floccosus), and the citrus blackfly (A. woglumi); the citrus mealybug (Planococous citri); three species of aphids: the green citrus aphid (Aphis spiraecola), the cotton or melon aphid (A. gossypii), and the brown citrus aphid (Toxoptera citricida), which is the vector of CTV (Stansly and Rogers, 2017); three species of root weevils: Diaprepes root weevil (Diaprepes abbreviatus), the blue-green citrus root weevil Pachnaeus litus, and the Northern citrus root weevil Pachnaeus opalus (Duncan et al., 2017a).

Species of mites that attack citrus include the pink citrus mite (Aculops pelekassi), the citrus rust mite (Phyllocoptruta oleivora), and the Texas citrus mite, Eutetranychus banksi (Rogers and Stansly, 2017).

Nematode species of major economic importance in Florida citrus include Tylenchulus semipenetrans, which causes “slow decline” of citrus, and the burrowing nematode Radopholus similis, which causes the “spreading decline” of citrus. Nematodes of limited economic importance include the sting nematode Belonolaimus longicaudatus, and two species of lesion nematode, Pratylenchus coffeae and P. brachyurus (Duncan et al., 2017b). For more information on Florida citrus pests and diseases, see the 2017–2018 Florida Citrus Production Guide (Rogers et al., 2017).

Citrus Production

For many years the United States was the third largest citrus producer in the world, after China and Brazil; however, Food and Agriculture Organization (FAO) data for 2015 (FAO, 2017a) indicate that the United States

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⁸ An infectious entity affecting plants, smaller than a virus and consisting only of nucleic acid without a protein coat.

is currently behind India in total citrus production. It now ranks fourth and is followed by Mexico and Spain (FAO, 2017b, Table 1, pp. 14–15). While citrus is produced by several countries around the world, orange juice production occurs mainly in Brazil and Florida, with Brazil as the largest orange juice exporter (UNCTAD, 2010).

The major citrus-producing states in the United States are Florida, California, Arizona, and Texas; smaller quantities of citrus are grown in other Sunbelt states⁹ and in Hawaii. During the 2016–2017 season in the United States, citrus was grown on 711,000 acres (288,000 hectares) with a total fruit yield of 7.8 million tons. Approximately half of the U.S. citrus produced is sold fresh, and the remainder is sold processed, mostly as juice, with California producing 85% of the fresh and Florida 77% of the processed citrus (USDA NASS, 2017).

Genetics, Breeding, and Biotechnology

The genetic complexity of the genus Citrus results in high levels of heterozygosity, and consequently many generations of breeding and selection are required to develop new cultivars having the combinations of genes that produce the desired fruit and juice quality, productivity, and stress and disease resistance traits that are required for the commercial fresh or processing markets. The long juvenility period (3 to 7 or more years) for citrus grown from seed, as is necessary in a breeding program, along with the large areas of land required to grow and evaluate trees, add to the challenges of citrus breeding. Breeding can be hampered also by the peculiar biology of some citrus types or cultivars; nucellar embryony or polyembryony is a condition in which embryos are produced from the seed nucellus, which is derived from the mother tree (Kepiro and Roose, 2007). Since these nucellar embryos grow more rapidly than the hybrid embryo produced from cross pollination, they crowd out the hybrid embryo, which does not develop further. In this case, even though a hybridization was made to produce a new genetic combination, the result is simply a reproduction of the original mother tree and provides no genetic advancement. While a great deal of breeding has been carried out in the United States and other major citrus-growing areas, most of the commercial cultivars currently grown were selected in the 19th century (Purseglove, 1968).

Genetic improvement through genetic engineering offers the potential of circumventing some of the major hurdles affecting conventional

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⁹ U.S. Sunbelt states include Alabama, Arizona, Florida, Georgia, Louisiana, Mississippi, New Mexico, South Carolina, Texas, roughly two-thirds of California (up to Greater Sacramento), and parts of Arkansas, Nevada, and North Carolina.

breeding and allows for the targeted improvement of existing cultivars, avoiding random assortment of genes and loss of the genetic integrity of the desired cultivar. Genetic engineering relies on transformation, which is the ability to insert a single gene or multiple genes into plant cells and to regenerate whole plants from the transformed cells. Transformation of citrus is specific to genus, species, and genotype and is limited to relatively few genotypes (Donmez et al., 2013). It has generally been undertaken with nucellar embryos, which are produced from the parent tree and are not hybrid. While these explants reproduce the original source tree, the time to fruiting is extended due to juvenility. Transformation rates are lower when using explants from mature trees but the time to fruiting for transgenic plants is reduced (Marutani-Hert et al., 2012). Commercially important cultivars that can be transformed include “Pineapple,” “Hamlin” and “Valencia” oranges, “Rio Red” grapefruit, “Eureka” lemon, and “Sucarri” sweet orange (Hu et al., 2016). Nevertheless, rates of transformation are quite low for many genotypes, generally less than 1% transformed plants per total number of explants (Hu et al., 2016). An alternative to the “conventional” genetic engineering technologies is the use of viral-based vectors for the expression of transgenes in citrus plants. This technology does not require the regeneration of plants from genetically transformed tissues but rather the infection of plants with an otherwise symptomless virus expressing a gene(s) for resistance to HLB (Folimonov et al., 2007; Agüero et al., 2012). Expression of the resistance factor may be translocated to all plant parts depending upon the virus, and infection may be initiated through grafting in the nursery or field. Application of gene-editing technologies in citrus should allow for genetic improvement of cultivars by deleting or adding a small number of base pairs in an existing gene that is responsible for a particular trait to be improved (Jia and Wang, 2014; Peng et al., 2017). Gene editing may also be used to add genes from the same species or other organisms as practiced through genetic engineering. Transformation is required for most gene-editing technologies.

To utilize many biotechnological improvement techniques, the identification of genes and gene function is required. The genomes of several citrus cultivars and species have been sequenced¹⁰ (Xu et al., 2013; Wu et al., 2014), 9 and the identification of genes is under way, a process that must take into account the large and complex genomes of citrus species and cultivars.

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¹⁰ Citrus genome database URL: https://www.citrusgenomedb.org. Accessed December 18, 2017.

HOST PLANT–PATHOGEN INTERACTIONS AND HOST PLANT DEFENSE MECHANISMS

The molecular interactions between host plants and pathogens determine whether the host will be susceptible, tolerant, or resistant. In the case of HLB, citrus host susceptibility is associated with the development of a variety of symptoms associated with HLB, including reduced fruit production and quality. Study of these interactions provides the ground for basic understanding of the disease and presents targets for control remedies (Wang and Trivedi, 2013; da Graça et al., 2016; Wang et al., 2017). After inoculation of phloem sieve tubes in the citrus host during feeding of the ACP, Ca. Liberibacters exploit phloem cellular processes for nutrient acquisition. Annotation of pathogen genomes has revealed hints about how these host nutrients are utilized, although experimental evidence for the dependence of those genes on host nutrients for pathogen viability have yet to be obtained. Ca. Liberibacter genomes carry genes encoding transporters and enzymes for metabolism of nucleotides, carbohydrates, amino acids, and lipids derived from host cells (Duan et al., 2009; Lin et al., 2011; Wulff et al., 2014).

Plant defense responses to pathogens are triggered by recognition of conserved “pathogen-associated molecular patterns” (PAMPs) by plant receptors, resulting in a partial reduction of pathogen growth. If the pathogen secretes virulence proteins that are recognized by resistance (R) proteins in the plant, a much stronger form of resistance will occur to arrest pathogen growth completely, making R genes the optimal source of crop disease resistance. Concerted screening efforts have not found evidence of R genes to HLB in any variety of citrus. However, there is substantial variation in the disease tolerance of different citrus varieties and rootstocks; “tolerance” is the ability of a plant to keep growing and producing fruit in the presence of infection. Tolerant citrus varieties have been found to accumulate a greatly reduced pathogen load compared with susceptible varieties (Albrecht and Bowman, 2012) and show less severe symptoms (Fan et al., 2012).

Known HLB-tolerant varieties have been a focus of citrus hybridization efforts, and study of tolerance mechanisms may lead to better strategies for screening and breeding for tolerance. Tolerance may be related to preformed pathogen barriers (such as structural and chemical defenses) or an increased ability to actively mount an effective PAMP-triggered immunity (PTI). However, compared with some other well-studied gram-negative bacterium–plant systems, basic knowledge about the interactions between Ca. Liberibacters and their hosts has lagged, largely due to the unculturability of the pathogen in vitro (Wang et al., 2017; Blaustein et al., 2018). CLas induces a variety of host hormonal and defense gene changes consistent with PTI (Kim et al., 2009; Pitino et al., 2017), and genomic analysis

indicates the presence of typical PAMP elements (Duan et al., 2009). One CLas-derived PAMP, CLas-flg22, is sufficient to trigger defenses in citrus and tobacco (Zou et al., 2012; Shi et al., 2018). CLas-flg22 is a conserved N-terminal 22–amino acid domain that triggers defense gene activation and physiological responses (callose deposition) when expressed in Nicotiana benthamiana cells; the activation of many citrus defense genes by flg22 is stronger in tolerant than in susceptible cultivars (Zou et al., 2012; Shi et al., 2018). Treatment of citrus with either CLas or CLas-flg22 caused distinct defense gene expression responses in tolerant and susceptible orange and grapefruit varieties, allowing the identification of candidate tolerance-associated genes (Wang et al., 2016; Shi et al., 2018). However, some defense mechanisms could actually harm the plant, resulting in phytotoxicity and symptom development or causing emission of volatile signals that attract ACP (Mann et al., 2012b; Pitino et al., 2017).

Plant defense mechanisms are typically suppressed by pathogen virulence proteins, and CLas appears to suppress defense responses in citrus (Nwugo et al., 2013a). Study of predicted pathogen virulence genes, revealed through genome sequencing, has provided clues to important components of the citrus defense response. Two predicted CLas-secreted proteins are very effective degraders of reactive oxygen species and salicylic acid, respectively, suggesting that these are critical defense signals in citrus, as they are in other plants (Jain et al., 2015; Li et al., 2017).

Citrus–Ca. Liberibacter interactions also involve effector proteins of bacterial origin that are predicted to modulate host cellular functions for the benefit of Ca. Liberibacter CLas multiplication and colonization of host phloem cells (Wang et al., 2017). Generally, microbial effectors play an important role in bacterial pathogenesis by restricting the host defense or interfering with host developmental processes in ways that benefit the pathogen (Jones and Dangl, 2006). Ca. Liberibacter genome sequences revealed the presence of substrate proteins derived from type I secretion systems (T1SSs)¹¹ and general secretory systems (Sec) in some Ca. Liberibacters, but genes for other secretion systems commonly found in other plant pathogenic bacteria are absent from the Ca. Liberibacter genomes sequenced to date (Duan et al., 2009; Lin et al., 2011; Fagen et al., 2014; Wulff et al., 2014). T1SS effector genes encoding serralysin and hemolysin have been identified from CLas isolates, but their role in host interactions remains to be experimentally determined (Duan et al., 2009). More than a hundred genes encoding substrates associated with Sec (i.e., containing a signal peptide for the Sec system) have been predicted for CLas strains (Prasad et al., 2016). Some predicted Sec-dependent effectors (SDEs) have

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¹¹ T1SS is a simple gram-negative bacterial secretion system that moves cytoplasmic molecules across all layers of the cell envelope and the periplasm in a single step.

been confirmed experimentally as substrates of Sec (Cong et al., 2012; Pitino et al., 2016). Although the role of SDEs in relation to HLB remains unclear, there is evidence that some could play an important virulence role. For example, Las5315 has been shown to be localized in the chloroplast and to induce callose deposition and trigger host cell death when expressed transiently in N. benthamiana (Pitino et al., 2016). Their role could be deciphered by in planta transient or transgenic expression in a plant, perhaps N. benthamiana or citrus.

Most importantly, the host plant responds to bacterial infection also at the transcriptional level. Citrus or citrus relatives, whether tolerant or susceptible to HLB, reprogram their transcriptomic network in response to Ca. Liberibacter infection. Response levels are generally stronger at earlier stages than later ones, and stronger in tolerant cultivars than in susceptible ones (Kim et al., 2009; Albrecht and Bowman, 2012; Fan et al., 2012; Martinelli et al., 2012, 2013; Aritua et al., 2013; Mafra et al., 2013; Yan et al., 2013; Zhao et al., 2013; Zheng and Zhao, 2013; Rawat et al., 2015, 2017; Xu et al., 2015; Fu et al., 2016; Wang et al., 2016; Zhong et al., 2016; Hu et al., 2017; Yu et al., 2017). The transcriptomic responses involve upregulated genes that can be categorized into a range of biological processes with which they are associated, such as those related to defense networks, photosynthesis and metabolism, hormone-mediated signaling networks, cell wall metabolism, and reduction/oxidation processes. Most defense or stress-response genes are upregulated in all genotypes of host plants at an early stage of infection (Mafra et al., 2013; Martinelli et al., 2013), but some, such as those involved in the mitogen-activated protein kinase (MAPK) signaling pathway, activation of peroxidases, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and POD4, and nucleotide binding site–leucine-rich repeat (NBS-LRR) type genes, are differentially upregulated in tolerant genotypes (Fan et al., 2012; Nwugo et al., 2013b; Hu et al., 2017; Yu et al., 2017).

Genes related to callose deposition in the phloem of infected plants (both susceptible and tolerant cultivars) are upregulated along with genes involved in starch biosynthesis, while genes involved in starch degradation are downregulated more strongly in susceptible than in tolerant genotypes (Boava et al., 2017). These findings are consistent with the hypothesis that callose deposition at the sieve pores interferes with the transport of photosynthetic products in phloem, enhancing the symptoms of HLB (Kim et al., 2009; Koh et al., 2012; Martinelli et al., 2013; Boava et al., 2017).

Certain genes reflecting the transcriptomic responses of host plants to Ca. Liberibacter infection can be confirmed at the proteomic level. For example, defense-related chitinase, miraculin-like proteins, Cu/Zn-SOD, and lipoxygenase are upregulated in CLas-infected sweet orange plant leaves (Fan et al., 2011). Evidence of upregulation of radical ion detoxification

proteins (e.g., glutathione-S-transferases) for tolerance to CLas has also been reported (Martinelli et al., 2016).

Finally, host–pathogen interactions in HLB also include host metabolic responses that may result from the manipulation of metabolic pathways by the pathogen for its benefit, or may result from host cellular function for defense reaction. One of the most obvious host responses to Ca. Liberibacter infection is an increase in total amino acid abundance, indicating benefits to both pathogen and host defense (Killiny and Hijaz, 2016; Killiny and Nehela, 2017). Other metabolites, such as terpenoids, appearing at higher levels in tolerant than in susceptible genotypes, may play an important antibacterial role, restricting pathogen growth, while other metabolites, present at lower levels, may restrict pathogen nutrient acquisition (Hijaz et al., 2013, 2016; Albrecht et al., 2016; Killiny and Nehela, 2017).

PATHOGEN–VECTOR INTERACTIONS

CLas is dependent upon the ACP to move between plant hosts and perhaps to contribute to its long-term survivability, since the bacterium replicates to some extent in the insect (Inoue et al., 2009; Ammar et al., 2016). Because CLas impacts ACP fitness it is a pathogen of both plant and insect. There is some evidence that CLas evolved first as an insect pathogen or symbiont, later becoming a plant pathogen (Gottwald, 2010). In the ACP, CLas must first be ingested and then translocated into the gut tissues. Subsequently the bacteria can replicate and systemically infect the insect, ultimately moving to the salivary tissues from which they can be expelled with salivary secretions into other host plants (Ammar et al., 2011; Kruse et al., 2017).

As early as the mid-1960s, it was reported that an as-yet-unknown agent associated with HLB was transmissible by psyllids (da Graça, 1991). By the late 1960s, ACP was confirmed as a principal vector of the greening agent. Long before ACP and CLas were identified in the United States, the general characteristics of CLas transmission by ACP were well described, e.g., the duration of acquisition, latent, retention, and inoculation periods (da Graça, 1991). Also well described were the propensity of late instar and adult ACP to acquire CLas on young flush tissue and the attraction of ACP to the yellow-green color of HLB-infected trees. Although early reports suggested that ACP nymphs were not important in the transmission of CLas (da Graça, 1991), more recent studies have shown that early instar nymphs acquire CLas more efficiently than adults and that transmission efficiency is higher in ACP reared on CLas-infected plants than in adults fed on CLas-infected plants (Inoue et al., 2009; Canale et al., 2017), although long feeding times by adults tend to negate these transmission efficiency differences (Pelz-Stelinski et al., 2010).

The multitude of studies on transmission of CLas by ACP done over the past five decades support the conclusion that the general parameters of transmission are similar to expectations for most arthropod-borne pathogens that circulate in their vectors; i.e., longer acquisition and inoculation times facilitate transmission efficiency, and there is an optimal latent period measured in days between acquisition and transmission (Grafton-Cardwell et al., 2013). Higher pathogen titer in the source tissue will enhance acquisition and transmission efficiency, and higher numbers of the bacteria inoculated to a host in a susceptible state will facilitate infection efficiency (Ukuda-Hosokawa et al., 2015). Details of CLas infection of ACP have remained elusive. Longer acquisition times result in higher CLas titer in insects, but maintenance of “infected” ACP on plants that are not hosts of CLas result in conflicting results that bacterial titers can either decrease (Pelz-Stelinski et al., 2010) or increase over time (Ammar et al., 2016). This discrepancy may be due to the age of the psyllids used in the study. Other studies also suggest that CLas replication may occur in nymphs but not in adults (Inoue et al., 2009); this phenomenon may be related to the immune response in ACP, which appears to be suppressed in nymphs (Ramsey et al., 2017). Recent work has shown that several CLas genes are expressed differentially when the bacteria are associated with plants versus when they are associated with ACP. While the bacteria may not replicate efficiently in all ages of psyllids, it is clear that the bacteria survive for long periods of time in the insect and can be vertically transmitted, albeit at a low frequency (Mann et al., 2011).

VECTOR–HOST INTERACTIONS

As its common name suggests, ACP is native to Asia, but both scientific and common names are misleading with respect to its host plant use patterns. Although all known host plant species belong to the subfamily Aurantioideae in the family Rutaceae, ACP infests species in at least 10 genera in the family in addition to Citrus (Grafton-Cardwell et al., 2013). Not all genera reported as host plants are equally suitable for oviposition, development, and adult feeding and reproduction, however. Outside the Aurantioideae, white sapote (Casimiroa edulis), in the subfamily Toddalioideae, was rejected by all ACP life stages in free-choice field studies (Westbrook et al., 2011). Studies of host plant preference and suitability both within the genus Citrus and across the Rutaceae have confirmed a strong link to leaf flush, irrespective of species or genotype (Ruan et al., 2015; Hall and Hentz, 2016). For example, orange jasmine (Murraya paniculata) flushes continuously and, relative to commercial citrus, supports more rapid ACP population growth (Tsai and Liu, 2000). Among plants that are distinctly less preferred is P. trifoliata (hardy or trifoliate orange),

possibly due to reduced rates of oviposition. Of the native Rutaceae species potentially at risk of colonization by ACP in its North American nonindigenous range, Choisya ternata, C. arizonica, and Helietta parvifolia all support ACP growth and reproduction (Sétamou et al., 2016) and may serve as potential host plants. By contrast, six other native rutaceous species failed to support oviposition, nymphal development, or adult survival.

Multimodal sensory inputs are important in host finding by the ACP. With respect to chemosensory stimuli, the preference for newly flushed leaves is mediated at least in part by responses to volatile signals. As in other psyllids (Park and Hardie, 2002), the olfactory system of ACP is relatively simple; glomeruli are absent from the antennal lobes. Perception of host plant odors is associated primarily with the olfactory neurons innervating the rhinarial plates¹² on adult antennae. More than three dozen volatile odorants from a diversity of chemical classes have been characterized from Citrus and Bergera species that are suitable hosts for development. Among possible attractive odorants, monoterpenes, esters, and aldehydes elicited strong activation of olfactory neurons (Coutinho-Abreu et al., 2014), as did acetic and formic acid, both of which are breakdown products of terpenoids (George et al., 2016). Olfaction also plays a role in behavior related to host plant rejection. Repellency of guava (Psidium guajava), a nonhost, is associated with the production of volatiles, including β-caryophyllene (Zaka et al., 2015; Alquézar et al., 2017). After settling, ACP assesses host plant suitability using gustatory cues. Mixtures of formic acid, acetic acid, and p-cymene can stimulate probing and salivary sheath formation (Lapointe et al., 2016).

Visual cues also influence host plant choice by ACP. The day-flying adults are positively phototactic and are attracted to yellow sticky traps (Sétamou et al., 2016). Walking behavior is elicited by wavelengths in the short-wave range of the spectrum and by vertically oriented polarized white light. Responses to UV light may account for dispersal out of the canopy and settling on foliage for feeding when flushing foliage is less abundant (Paris et al., 2017). Responses to visual cues can change in the presence of either olfactory or gustatory chemical stimuli (Patt et al., 2011), although under field conditions in urban environments in California combining olfactory and color stimuli did not enhance capture rates over traps lacking any lures (Godfrey et al., 2013). To some extent, host plant location and assessment by adult ACP are influenced by previous developmental and adult experience. Under certain circumstances, ovipositing females display a preference for the host plant species on which they develop but these preferences can shift after exposure to an alternative host plant 1 or 2 days later (Stockton et al., 2016, 2017b).

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¹² The lower part of the clypeus, which is a plate on the anterior median aspect of the head.

Adult ACPs are attracted to yellow and yellowish green colors that mimic the reflectance spectra of their rutaceaous host plants. The recent identification of methyl salicylate (MeSA) as an attractive host plant volatile (Mann et al., 2012a) has been investigated as a means of improving the deployment of sticky traps for monitoring ACP, but this approach has met with only limited utility in the development of attract-and-kill technologies because of the widespread distribution of diseased trees in selected areas (Yan et al., 2014). CLas infection of the citrus tree further induces the release of MeSA, which renders infected plants more attractive than uninfected plants. However, infected trees are less suitable than uninfected plants as hosts for development of ACP, and the tendency of psyllids to leave infected plants shortly after pathogen acquisition may promote the spread of the pathogen.

DIAGNOSTICS

Accurate, sensitive, and cost-effective means of detecting CLas infections in both citrus trees and ACP are of paramount importance to the management of HLB. There is an extensive literature describing various techniques to detect CLas in plant and insect tissues (Valdes et al., 2016). Because CLas has not yet been cultured outside its hosts, there are no methods to enrich the bacteria from selected host tissues to facilitate detection by molecular, serological, or other means. A quantitative polymerase chain reaction (qPCR)-based assay (Li et al., 2006) for amplifying CLas 16S RNA has become the standard assay accepted by many laboratories and, more importantly, by regulatory agencies to provide an initial determination of CLas infection. This is followed by conventional PCR assays and DNA sequencing for final verification. Although many reports have been published in the past decade on other methods to detect CLas in plant and insect tissues (Valdes et al., 2016; Warghane et al., 2017), none of the mechanistically similar technologies (e.g., digital PCR, immunoblots, LAMP, or loop-mediated isothermal amplification, CANARY or Cellular Analysis and Notification of Antigen Risks and Yields) have proven to be more sensitive than qPCR. Furthermore, since qPCR can detect as little as one copy of bacterial DNA the issue for CLas detection is not the sensitivity of bacterial detection but rather the uneven spatial and temporal distribution of the pathogen in trees and insects (Tatineni et al., 2008; Li et al., 2009; Kunta et al., 2014; Louzada et al., 2016).

Further exacerbating the problems with the conventional diagnostics mentioned above is that there is a long latent period between inoculation of the plant and multiplication of the bacteria to detectable levels (Manjunath et al., 2008; Gottwald, 2010; Chiyaka et al., 2012). There are perhaps some parallels with movement and distribution of phloem-limited viruses, but it

is unclear if any studies have addressed this question. Several studies have shown that conventional detection of CLas in trees lags behind the time of inoculation by several weeks to months depending on the size and type of tree (Bové, 2006; Gottwald, 2010). More importantly, the tree is a source of bacteria for ACP vectors long before CLas can be detected.

HLB MANAGEMENT

The two basic management strategies for any vector-borne pathogen transmitted in a circulative manner are (1) to reduce the number of vectors available to transmit the pathogen and (2) to reduce the amount of inoculum available to the vectors. While considerable effort has gone into controlling ACP it seems that less effort has been devoted to the removal of inoculum sources, i.e., infected trees, even though research indicates that removal of infected trees can slow epidemics. The effectiveness at slowing and managing the incidence of HLB as well as that of other diseases having similar etiologies, using these two basic strategies, is well documented and supported (Belasque et al., 2010; Bassanezi et al., 2013a,b; Ayres et al., 2015; Bergamin Filho et al., 2016). This strategy, however, works only if the disease epidemics are in the early stages, i.e., when the level of inoculum is relatively low and identification and removal of infected plants does not compromise the ability of the producer to realize an acceptable level of economic return. This strategy may still be important in California, and possibly in Texas and Arizona, where the reported level of HLB in commercial citrus orchards is still low. However, optimization of the strategy will depend on the validation and acceptance of early detection technologies so that infected trees can be identified and removed prior to becoming sources of inoculum for ACP. Infected tree removal and vector control were the primary eradication strategies early on in Florida, prior to the realization that significant numbers of trees were infected and serving as sources of inoculum while still asymptomatic. Currently, the incidence of HLB in Florida citrus has reached or is approaching 100% and removal of inoculum on individual groves is no longer an option.

Vector control remains an important component of HLB management in Florida, although any type of effective HLB management will depend on area-wide implementation as well as cooperative and sustained participation by all citrus production operations. Citrus Health Management Areas (CHMAs) were established in Florida with the goal of regional participation in insecticide applications for ACP control. These have been only somewhat effective due to incomplete grower participation in some areas (Singerman et al., 2017). A CHMA-like approach has been effective in Brazil, but a component of their effectiveness has been continuous removal of infected trees and complete eradication of abandoned groves

and alternative hosts (Belasque et al., 2010). Timely and effective disposition of abandoned citrus groves and infected backyard trees in urban areas near commercial citrus production areas has not been a practice in Florida, and these pathogen sources continue to add to the disease pressure on all Florida citrus (Tiwari et al., 2010). Models predict that reducing ACP populations during critical times can reduce spread (Lee et al., 2015), but even with 100% participation in CHMAs, ACP population management in managed groves alone would be unlikely to significantly slow HLB progression due to the refugia for ACP and CLas provided by abandoned groves and backyard trees. ACP repellents can also slow and prevent transmission of CLas by immigrating insects. One promising strategy for protecting young transplants is the use of reflective mulch (Croxton and Stansly, 2014), which has been used in the management of several insect-borne plant viruses in other crop systems (Budnik et al., 1996; Abou-Jawdah et al., 2000; Antignus, 2000). An additional benefit of using reflective mulch as ground cover was increased citrus tree growth rate (Croxton and Stansly, 2014).

Other cultural control efforts have focused on the use of thermotherapy to reduce or eliminate CLas infection in trees. While thermotherapy has been successful at reducing CLas titer in small trees growing in the greenhouse (Fan et al., 2016), results with mature trees in commercial grove settings have been mixed (Yang et al., 2016; Doud et al., 2017).

Historically, Florida citrus groves have not been intensively managed with respect to nutrition and water, such that trees are continually exposed to some level of stress. Attacks of insects and pathogens, either direct or opportunistic, contribute to additional stress that affects fruit yields and quality (Ashraf et al., 2014). A growing trend in Florida is the manipulation of nutrition and irrigation regimes to reduce the effects of HLB on tree health, fruit production, and fruit quality. Although there is little peer-reviewed literature to support these actions by many growers (Kadyampakeni et al., 2015; Morgan et al., 2016; Hamido et al., 2017; Plotto et al., 2017), the general sense is that trees under any type of stress are less able to resist CLas infection and ACP infestation. If stress can be reduced by ensuring optimal irrigation and nutrition, the trees may show less severe disease symptoms, including milder effects on fruit production and yield. While some growers have reported milder disease symptoms, increased fruit yields, and improved fruit quality with fertigation, controlled research findings have been mixed (Gottwald et al., 2012; Stansly et al., 2014; Kadyampakeni et al., 2015; Morgan et al., 2016; Hamido et al., 2017; Plotto et al., 2017; Rouse et al., 2017; Tansey et al., 2017). The fact that effects of optimal nutrition and water are apparent only after 2 to 3 years of ideal management may explain why some of the early, shorter studies found no benefits (Plotto et al., 2017).

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