Tolerance of Trifoliate Citrus Rootstock Hybrids to Candidatus Liberibacter Asiaticus

Scientia Horticulturae 147 (2012) 71–80

Contents lists available at SciVerse ScienceDirect

Scientia Horticulturae

journa l homepage: www.elsevier.com/locate/scihorti

Tolerance of trifoliate citrus rootstock hybrids to Candidatus Liberibacter asiaticus

∗

Ute Albrecht, Kim D. Bowman

U.S. Horticultural Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, 2001 South Rock Road, Fort Pierce, FL 34945, USA

a r t i c l e i n f o a b s t r a c t

Article history: Candidatus Liberibacter asiaticus (Las) is the suspected causal agent of Huanglongbing (HLB) in Florida

Received 22 May 2012

and other citrus producing countries around the world. Although little resistance to HLB is found within

Received in revised form 29 August 2012

commercial citrus varieties, tolerance has been reported for some varieties that are commonly used as

Accepted 31 August 2012

rootstocks. This study assesses the response of eight different rootstock varieties, which include the Cit-

rus × Poncirus trifoliata hybrids Carrizo citrange, US-802, US-812, US-897, and US-942, Benecke trifoliate

Keywords:

orange, Volkamer lemon, and Cleopatra mandarin, to Las under controlled conditions in the greenhouse.

Citrus disease

The percentage of plants detected positive for Las did not exceed 7% in the genotypes Benecke, Carrizo,

Citrus greening

US-802, US-942, and Volkamer during the ﬁrst 6 months after graft-inoculation (mai). Pruning resulted

Disease resistance

in much increased numbers of plants with detectable Las levels and induced disease symptoms in pre-

Poncirus trifoliata

Rootstock viously asymptomatic plants. At 12 mai, percentages of Las-positive plants were 91–96% for US-802,

US-812, Cleopatra, and Volkamer, and 70–78% for US-897 and US-942. Lowest percentages (44–52%) were

observed for Benecke and Carrizo. The number of Las genomes per g of leaf tissue in Las-positive plants

5 6

was considerably lower in Benecke and US-897 seedlings with 4.5 × 10 and 1.6 × 10 compared with

7 6

Cleopatra (1.0 10 ), while numbers for the other genotypes ranged from 3.1–4.9 × 10 . At 12 mai, foliar

disease symptoms, though variable in extent, were prominent in most genotypes, except US-897. Shoot

masses were signiﬁcantly reduced in Cleopatra, Benecke, US-802, and Volkamer seedlings in response to

Las at this time. Continued observation until 18 mai found no signiﬁcant growth reductions in Benecke

seedlings. Our results suggest classifying Carrizo, US-897, and US-942 as tolerant, US-802, US-812, and

Volkamer lemon as moderately tolerant, and Cleopatra mandarin as susceptible to Las. Despite irregular

growth, low rates of infection and low Las numbers indicate some resistance of Benecke to Las. Addi-

tional greenhouse experiments and field observations confirmed findings for US-802, US-897, US-942,

and Cleopatra, although results for US-802 were more variable.

1. Introduction China, and is now found in most citrus-growing areas worldwide,

including Asia, Saudi Arabia, Africa, and the Americas (Bové, 2006;

Candidatus Liberibacter spp. are the suspected causal agents Gottwald, 2010). The disease was found in Florida in 2005 (Halbert,

of Huanglongbing (HLB), a destructive disease of citrus which 2005), 1 year after its ﬁrst discovery in Sao Paula, Brazil (Teixeira

threatens citrus production in Florida and other citrus-producing et al., 2005).

countries. Three Candidatus Liberibacter spp. are associated with The Chinese name Huanglongbing refers to the appearance of

HLB: Ca. L. asiaticus, Ca. L. africanus (Jagoueix et al., 1994) and yellow shoots in the canopy of affected trees, which are the result

Ca. L. americanus (Teixeira et al., 2005). The ranking of Candida- of an asymmetric blotchy mottling of leaves or severe chloro-

tus was assigned due to the difﬁculty of obtaining the pathogen sis, similar to zinc- or other nutrient deﬁciencies (McClean and

in pure culture (Davis et al., 2008; Sechler et al., 2009). Liberib- Schwarz, 1970). Fruit of affected trees become misshapen, develop

acters are gram-negative bacteria (Garnier et al., 1984) which are an undesirable taste and drop prematurely, thus generating sub-

transmitted to the phloem by sap-sucking insects of the family Psyl- stantial losses to citrus growers in affected areas. Crop losses on

lidae. In Florida and most other countries, HLB is associated with juice orange production in Florida during the past 5 years because

Ca. L. asiaticus (Las) vectored by the Asian citrus psyllid Diapho- of HLB are estimated to be more than 20% (Hodges and Spreen,

rina citri Kuwayama (Halbert and Manjunath, 2004; Halbert, 2005). 2011). HLB symptom development appears to be associated with

HLB probably originated in India in the 1700s before spreading to phloem collapse and blockage of the translocation stream as a

result of infection with liberibacters (Schneider, 1968; Achor et al.,

2010). In addition, infection induces major changes in carbohydrate

∗ metabolism, phytohormone metabolism and other metabolic path-

Corresponding author. Tel.: +1 772 462 5920; fax: +1 772 462 5986.

E-mail address: [email protected] (K.D. Bowman). ways (Albrecht and Bowman, 2008, 2012; Kim et al., 2009; Fan et al.,

72 U. Albrecht, K.D. Bowman / Scientia Horticulturae 147 (2012) 71–80

2010, 2011; Rosales and Burns, 2011). Current management strate- pieces from infected greenhouse-grown ‘Valencia’ orange plants,

gies for HLB consist of planting disease-free trees, vector insect PCR-positive for Ca. L. asiaticus (Las) and symptomatic for HLB, onto

control in combination with nutritional applications, and removal each plant. Twelve plants of each genotype were mock-inoculated

of infected trees. with disease-free tissue pieces obtained from healthy greenhouse-

HLB affects all citrus species and relatives, and commercial vari- grown ‘Valencia’ orange plants to be used as non-infected controls.

eties such as sweet oranges (Citrus sinensis L.), mandarins (Citrus

reticulata Blanco) and tangelos (hybrids of C. reticulata) are most

2.1.2. Experiment 2

susceptible (McClean and Schwarz, 1970; Nariani, 1981; Lopes

Thirty-six greenhouse-grown 16-month-old seedlings of the

and Frare, 2008; Miyakawa, 1980; Miyakawa and Zhao, 1990;

genotypes Benecke trifoliate (P. trifoliata (L.) Raf.), Carrizo citrange

Folimonova et al., 2009). Tolerance to HLB has been reported for

× ×

(C. sinensis P. trifoliata), US-802, US-812 (C. reticulata ‘Sunki’ P.

some cultivars commonly used as rootstocks, particularly trifoli-

trifoliata ‘Benecke’), US-897, US-942 (C. reticulata ‘Sunki’ P. trifoli-

ate orange (Poncirus trifoliata L. Raf.) and some of its hybrids. In

ata ‘Flying Dragon’) and Volkamer lemon (Citrus volkameriana Ten.

South Africa, McClean and Schwarz (1970) did not ﬁnd any well-

& Pasq.) and 29 greenhouse-grown 16-month-old Cleopatra man-

deﬁned disease symptoms in P. trifoliata after graft-inoculation

darin seedlings were used. Plants were inoculated in July 2010 as

with diseased bud tissue. Similarly, Miyakawa (1980) found no dis-

described above, but using 3 bark- or bud pieces per plant. Nine

tinct HLB symptoms for P. trifoliata in Taiwan. However, moderate

plants of each genotype were mock-inoculated with disease-free

to severe disease symptoms were observed for trifoliate orange

tissue pieces and 27 plants were inoculated with infected tissue. For

and its hybrids Carrizo citrange and Troyer citrange (C. sinen-

Cleopatra mandarin, 6 plants were mock-inoculated and 23 plants

sis L. Osbeck × P. trifoliata) by Nariani (1981) in India. A study in

were inoculated with infected tissue.

Florida reported inconsistent results for greenhouse-grown P. tri-

All inoculations were performed in groups containing one plant

foliata seedlings after graft-inoculation with Las-infected budwood

per genotype to ensure that different genotypes received tissue

(Folimonova et al., 2009). In the same study, Carrizo citrange was

pieces from the same source.

classiﬁed as tolerant to HLB based on the absence of foliar dis-

ease symptoms and healthy growth of infected plants. A recent

2.1.3. Experiment 3

study conducted in our laboratory identiﬁed the trifoliate hybrid

Thirty 18-month-old greenhouse-grown plants with US-942

US-897 (C. reticulata × P. trifoliata) as tolerant to HLB (Albrecht and

scion, generated from etiolated internode stem segments using

Bowman, 2011). Transcriptional analysis of US-897 discovered a

standard in vitro plant regeneration procedures (Edriss and Burger,

number of genes possibly associated with tolerance to Las (Albrecht

1984; Zou et al., 2008) and micro-grafted onto decapitated epi-

and Bowman, 2012).

cotyls of seed-grown 3–5-week-old Volkamer lemon rootstock via

The rootstock is an important component of commercially

wedge-grafting, were used. Plants were inoculated in July 2010 as

grown citrus trees, and may determine success or failure of a cit-

described for experiment 2. Ten plants were mock-inoculated with

rus operation (Castle, 2010). In addition to the desired effect on

Las-free tissue pieces and 20 plants were inoculated with infected

scion vigor, fruit size, fruit quality, and yield, rootstock selection is

tissue.

based on tolerance to different environmental conditions and resis-

tance to pests and diseases. The USDA citrus breeding program has

2.1.4. Experiment 4

been developing and evaluating new citrus varieties since 1893 and

Twenty-nine 18- to 24-month-old greenhouse-grown plants

several new U.S. rootstock selections have been released in recent

with US-812 scion, in vitro-generated and micro-grafted onto

years (Bowman, 2001, 2007a,b, 2010). Although a ﬁeld study con-

Volkamer lemon as described for experiment 3, were used. Plants

ducted in our laboratory found that rootstock did not induce high

were inoculated in September 2010 as described for experiment 2.

levels of resistance to HLB in the scion, differences in disease symp-

Nine plants were inoculated with disease-free tissue pieces and 20

tom expression, canopy damage, stem growth, and fruit quality

plants were inoculated with infected tissue.

were detected on some rootstock selections (Albrecht et al., 2012).

Plants were arranged randomly on the greenhouse benches and

Studies in India and South Africa also found improved tolerance to

◦

kept under natural light conditions at a temperature of 21–28 C.

HLB in different scion/rootstock combinations (Nariani, 1981; Van

Plants were irrigated as needed and fertilized every 3 weeks using

Vuuren and Moll, 1985).

a water-soluble fertilizer mix, 20N-10P-20K (Peters Professional,

The objective of this study was to assess the response and the

The Scotts Company, Marysville, OH). Plants were pruned imme-

degree of tolerance to Las in several trifoliate hybrid rootstock

diately after graft-inoculation to promote new leaf growth and

selections and other rootstocks important to citrus production in

enhance HLB disease symptom development. Plants were addi-

Florida. Knowledge of the degree of tolerance to HLB is essential

tionally pruned 30 weeks after inoculation (experiment 1) or 6

for resistance breeding and the development of alternative hor-

months after inoculation (experiments 2–4). Plants were treated

ticultural practices, such as the use of double or auxiliary scions

with insecticides as needed. Insecticide treatments included three

(Stuchi and Girardi, 2010) or interstocks (Shokrollah et al., 2011)

foliar applications of the miticide Kelthane (Kelthane MF, Rohm &

which may mitigate the destructive effects of this disease.

Haas, Philadelphia, PA) in October/November 2010 at a concentra-

tion of 1.5 mL/L.

2. Materials and methods

2.1.5. Field-grown trees

2.1. Plant material Seventeen trees with US-802 scion and 20 trees each with US-

897 and US-942 scions, located at the USHRL-USDA Farm in St. Lucie

2.1.1. Experiment 1 County, Florida, were available for study. The trees, which are on

Forty greenhouse-grown 9-month-old US-802 (Citrus grandis different rootstock varieties, were planted in November 2004 and

(L.) Osb. ‘Siamese pummelo’ P. trifoliata ‘Gotha Road’ trifoli- are located in an area heavily affected by HLB. Trees were irri-

ate orange), US-897 (C. reticulata ‘Cleopatra’ P. trifoliata ‘Flying gated by micro sprinklers three times a week in the absence of

Dragon’) and Cleopatra mandarin (C. reticulata Blanco) seedlings adequate rainfall. Fertilizer and pesticides were applied season-

were used. Twenty-eight plants of each genotype were inoculated ally or as needed, but did not include increased foliar nutritional

in November 2009 by grafting 2 bark- or bud-pieces and 2 leaf applications (http://edis.ifas.uﬂ.edu/hs1165) or aggressive psyllid

U. Albrecht, K.D. Bowman / Scientia Horticulturae 147 (2012) 71–80 73

Table 1

Percentage of plants from experiment 1 PCR-positive for Ca. L. asiaticus (Las) and average number of Las genomes 6 to 48 weeks after inoculation (wai).

Weeks after inoculation

6 12 18 24 30 48

Percentage of PCR-positive plants

US-802 0 0 0 0 0 75

US-897 0 4 14 32 43 100

Cleopatra mandarin 0 68 71 71 79 100

Number of Las genomes (PCR-positive plants)

US-802 – – – – – 431,208

US-897 – 19,641 3,720,408 1,952,757 1,491,682 1,271,584

Cleopatra mandarin – 2,188,949 10,897,119 12,847,547 16,186,076 4,879,260

Number of Las genomes (Las-inoculated plants)

US-802 13 22 15 15 34 35,927

US-897 12 17 89 1,628 2,522 1,271,584

Cleopatra mandarin 13 121,307 700,679 712,049 1,278,396 4,897,260

Numbers of Las genomes are expressed per gram of plant tissue. Plants were pruned after sample collection at 30 wai. A total of 28 plants per genotype were analyzed.

control as currently practiced in many HLB-affected commercial 2.5. Growth performance

citrus groves in Florida.

Stem diameters were measured at 7 cm (experiment 1) or 8 cm

(experiment 2) above soil level using a digital caliper. Scion- and

2.2. PCR detection of Las in leaves

rootstock diameters of plants from experiments 3 and 4 were mea-

sured at 2 cm above and below the graft union. All measurements

Depending on leaf size, three to six leaves were collected from

occurred below the inoculation site. Growth was expressed as per-

each plant at 6, 12, 18, 24, 30, and 48 weeks after inoculation (exper-

cent increase of mean stem diameters from the beginning to the

iment 1), at 3, 6, 9, and 12 months after inoculation (experiment 2)

end of the study. At the end of experiments 2, 3 and 4, shoots grown

and at 2, 4, 6, 9, and 12 months after inoculation (experiments 3

above the pruning site were severed and weighed on a digital scale.

and 4). In addition, leaves were collected from Benecke-, Carrizo-

Growth was assessed at 48 weeks (experiment 1) or at 12 months

and US-942 seedlings of experiment 2 18 months after inoculation.

after inoculation (experiments 2, 3 and 4). Benecke-, Carrizo- and

Five to ten leaves were collected from each ﬁeld tree in September

US-942 seedlings from experiment 2 were additionally assessed at

2007, June 2009 and August 2011. Petioles and parts of the midrib

18 months after inoculation.

were severed from leaves and ground in liquid nitrogen with a

mortar and pestle. One hundred mg of ground tissue was used for

DNA extraction. DNA was extracted using the Plant DNeasy Mini

3. Results

Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s

instructions, yielding 20–30 ng DNA per extraction. For detection

3.1. Experiment 1

of Las, real-time PCR assays were performed using primers HLBas

and HLBr and probe HLBp developed by Li et al. (2006). For nor-

3.1.1. Percentage of PCR-positive plants and number of Las

malization, all samples were assayed using primers COXf and COXr genomes

and probe COXp (Li et al., 2006). Ampliﬁcations were performed

At 48 weeks after inoculation (wai), 75% of US-802 seedlings

over 40 cycles using an ABI 7500 real-time PCR system (Applied

were identiﬁed as PCR-positive for Las and the average number

Biosystems, Foster City, CA, USA) and the QuantiTect Probe PCR Kit 5

of Las genomes in PCR-positive plants was 4.3 × 10 (Table 1). No

(Qiagen) according to the manufacturer’s instructions. All reactions

PCR-positive US-802 plants were detected before that time. The

were carried out in a 20 ␮l reaction volume using 5 ␮l DNA. Plants

percentage of PCR-positive US-897 seedlings ranged from 4% at

were considered PCR-positive when normalized Ct -values were

LAS 12 wai to 100% at 48 wai with average Las genome numbers of

≤33. 4 6

2.0 × 10 to 3.7 × 10 . The average Las genome number for US-897

plants at the end of the experiment was 1.3 × 10 . At 12 wai, 68%

2.3. Quantiﬁcation of Las of Cleopatra seedlings were PCR-positive for Las and contained an

average of 2.2 × 10 genomes. Incidence of detection increased to

For quantiﬁcation of Las, CtLAS-values were converted to copy 100% at 48 wai, at which time an average of 4.9 10 Las genomes

numbers of Las genomes based on a standard curve created in was measured in the plants. The number of genomes calculated for

our laboratory: y = 12.34 − 0.32x, where y = log of Las copy num- all Las-inoculated US-802 seedlings at the end of the experiment

ber and x = normalized CtLAS-value. Ct-values not determined after was 3.6 10 , and thus 35-fold lower compared with US-897 and

40 cycles were assigned a value of 41. Since three copies of the 136-fold lower compared with Cleopatra seedlings.

16s rDNA gene are present in the Las genome (Duan et al., 2009),

copy numbers were divided by three and data were expressed as

3.1.2. Foliar disease expression

numbers of Las genomes per gram of plant tissue.

Leaves of Las-inoculated US-897 seedlings did not look different

than leaves from mock-inoculated US-897 seedlings throughout

2.4. Foliar disease expression the study. No foliar HLB symptoms were observed for US-802

seedlings from 6 to 30 wai. At 48 wai, following cut-back of the

Plants were examined for chlorosis, blotchy mottle or other seedlings, 32% of all Las-inoculated US-802 seedlings displayed a

foliar abnormalities presumably associated with HLB at the time of light to moderate blotchy mottling of leaves. Blotchy mottle was

leaf collection. Evaluation was hampered in experiments 2, 3 and 4 generally restricted to less than 25% of leaves of affected plants,

as a result of miticide treatment, which caused a chlorotic blotch- but in some plants extended to 50% or more leaves. Not all US-802

ing in many of the plants, but was eliminated after the pruning at seedlings with blotchy mottle were PCR-positive for Las. Mock-

six months after inoculation. inoculated US-802 seedlings did not display any blotchy mottle.

74 U. Albrecht, K.D. Bowman / Scientia Horticulturae 147 (2012) 71–80

140 Table 2

P = 0.0007 Percentage of plants from experiment 2 PCR-positive for Ca. L. asiaticus (Las) and

120 average number of Las genomes 3–12 months after inoculation (mai).

100 Genotype Months after inoculation

36 9 12

Ctrl Percentage of PCR-positive plants

Benecke 0 4 22 52

60 Las

Carrizo citrange 4 4 37 44

40 US-802 4 4 93 93

US-812 7 15 74 96

US-897 26 26 74 78

Stem diameter growth (%) 20

US-942 0 0 52 70

Cleopatra mandarin 52 56 78 91

Volkamer lemon 4 7 78 93

US-802 US-897 Cleopatra a

Number of Las genomes (PCR-positive plants)

Benecke – 429,678 47,929 445,679

Fig. 1. Stem diameter growth (bottom) of mock-inoculated (Ctrl) and Ca. L. asiaticus

Carrizo citrange 344,562 15,462,763 223,125 4,878,381

(Las)-inoculated citrus seedlings from experiment 1 at 48 weeks after inoculation. P-

US-802 201,578 133,791 111,639 3,338,374

values for signiﬁcant (P < 0.05) differences between mock- and Las-inoculated plants

US-812 157,525 1,289,077 410,975 3,794,556

are indicated. Vertical bars are standard errors of the mean. Twelve mock-inoculated

US-897 757,358 773,526 91,241 1,580,996

and 28 Las-inoculated plants per genotype were analyzed. US-942 – – 111,059 3,146,243

Cleopatra mandarin 2,015,634 6,002,194 991,873 10,406,145

Volkamer lemon 6,153,063 2,395,087 439,456 4,946,469

Leaf chlorosis was observed in 68% of Las-inoculated Cleopatra

Number of Las genomes (Las-inoculated plants)

seedlings at 18 wai and at 24 wai. At 30 wai, chlorosis increased

Benecke 33 17 163 8471

in severity and was observed on the majority of leaves in 75% of Carrizo citrange 31 38 584 23,146

US-802 27 19 64,849 1,538,006

Las-inoculated Cleopatra plants. All Cleopatra seedlings displaying

US-812 26 237 33,583 2,796,965

foliar HLB symptoms were PCR-positive for Las. At 48 wai, follow-

US-897 1382 355 9170 171,053

ing cut-back of the plants, 32% of Las-inoculated Cleopatra seedlings

US-942 19 11 2648 289,116

displayed yellowing of the veins and most (64%) plants were visibly Cleopatra mandarin 18,982 57,441 122,172 4,786,821

stunted. Mock-inoculated Cleopatra seedlings did not display any Volkamer lemon 85 39 72,280 2,864,511

foliar abnormalities or stunting throughout the experiment. a

Numbers of Las genomes are expressed per gram plant tissue. Plants were

pruned after sample collection at 6 mai. A total of 27 plants per genotype were

analyzed, except for Cleopatra where only 23 plants were analyzed.

3.1.3. Growth performance

Forty-eight weeks after the start of the experiment, stem diam-

eters of US-802 and US-897 seedlings increased 93% and 30% increased to 4.8 × 10 at 48 wai. At 12 mai, Las numbers measured

on average, respectively, and growth did not differ signiﬁcantly for the other genotypes were 565-fold lower (Benecke), 207-fold

(P > 0.05) between mock- and Las-inoculated plants (Fig. 1). Stem lower (Carrizo), 17- to 28-fold lower (US-897and US-942), and 2-

diameters of Las-inoculated Cleopatra seedlings increased 83% on to 3-fold lower (US-812, Volkamer and US-802). Las numbers for all

average which was signiﬁcantly less than observed for mock- inoculated Benecke, Carrizo and US-942 seedlings at 18 mai were

5 5 4

inoculated controls which increased 119% in diameter. 4.6 × 10 , 1.4 × 10 and 3.7 × 10 , respectively.

3.2. Experiment 2 3.2.2. Foliar disease expression

At 3 mai, 43% of Las-inoculated Cleopatra seedlings displayed

3.2.1. Percentage of PCR-positive plants mildly chlorotic and/or blotchy mottled leaves (Table 3). Blotchy

Less than 8% of seedlings were detected PCR-positive for Las mottle was also observed on one Volkamer seedling. None of

in the genotypes Benecke, Carrizo, US-802, US-942, and Volkamer the other genotypes displayed foliar abnormalities at this time.

within the ﬁrst 6 months after inoculation (Table 2). The high- Chlorosis and blotchy mottle of leaves intensiﬁed in Cleopatra

est percentage (52–56%) of Las-positive plants was observed for seedlings and were visible on 52% of plants at 6 mai. The extent

Cleopatra seedlings during this time, whereas incidence of detec- of foliar disease symptoms in affected Cleopatra seedlings varied

tion for US-812 and US-897 ranged from 7% to 26%. At 9 mai, from 25% to 75% of leaves on symptomatic plants. Blotchy mot-

following pruning of the seedlings, the percentage of PCR-positive tle was also detected on one Carrizo seedling and two Volkamer

plants increased considerably in all genotypes and ranged from 22% seedlings at 6 mai. Las-inoculated plants of the other genotypes did

in Benecke to 93% in US-802. At 12 mai, the highest percentage not show a leaf phenotype different from mock-inoculated plants

of PCR-positive plants was detected in US-812 (96%), followed by at this time point. However, miticide treatments, which occurred

US-802, Volkamer and Cleopatra (91–93%) and US-897 and US-942 3–4 months after inoculation, resulted in leaf damage in a large

(70–78%). The lowest percentage was observed for Benecke and number of plants, hampering foliar disease symptom assessment.

Carrizo and was 52% and 44%, respectively. The average number of At 9 mai, following pruning, mild chlorosis was observed in 39% of

Las genomes in PCR-positive plants at 12 mai was different between Las-inoculated Cleopatra seedlings and in one Volkamer seedling.

genotypes and was lowest in Benecke (4.5 × 10 ) and US-897 Benecke seedlings displayed chlorosis and necrosis of leaves

6 7

(1.6 × 10 ) and highest in Cleopatra (1.0 × 10 ). Genome numbers throughout the experiment and independent of inoculation with

of PCR-positive US-942, US-802, US-812, Carrizo, and Volkamer Las. No foliar HLB symptoms were observed in the other genotypes

6 6

ranged from 3.1 10 to 4.9 × 10 at this time, which is 2- to 3- at 9 mai. At 12 mai, the percentage of seedlings with foliar disease

fold lower compared with PCR-positive Cleopatra seedlings. PCR symptoms ranged from 26% in Carrizo to 81% and 87% in Volkamer

analysis of leaves collected at 18 mai detected Las in 96% of Benecke and Cleopatra. No foliar disease symptoms were observed on

seedlings, in 76% of Carrizo seedlings and in 70% of US-942 seedlings US-897 plants. Foliar evaluation of Benecke was impossible at that

5 6 6

and average Las numbers were 8.3 × 10 , 1.9 × 10 and 1.1 × 10 , time due to extensive leaf drop observed in most mock- and Las-

respectively. The average number of Las genomes calculated for all inoculated plants. Foliar HLB symptoms detected for Cleopatra and

inoculated plants at 3 mai was highest (1.9 × 10 ) in Cleopatra and Volkamer at 12 mai were chlorosis, which in Cleopatra was often

U. Albrecht, K.D. Bowman / Scientia Horticulturae 147 (2012) 71–80 75

Table 3

Percentage of seedlings from experiment 2 with foliar HLB symptoms and symptom type 12 months after inoculation (mai).

Genotype Percentage of symptomatic plants Symptom type

3 mai 6 mai 9 mai 12 mai

Benecke 0 0 0 n/a –

Carrizo citrange 0 4 0 26 Blotchy mottle

US-802 0 0 0 37 Blotchy mottle

US-812 0 0 0 44 Blotchy mottle

US-897 0 0 0 0 –

US-942 0 0 0 44 Leaf curl, reduced leaf size, chlorosis, necrosis

Cleopatra mandarin 43 52 39 87 Chlorosis, reduced leaf size, blotchy mottle

Volkamer lemon 4 7 4 81 Chlorosis, blotchy mottle

HLB symptoms assessment at this time was hampered due to damage from miticide treatment.

Due to extensive leaf drop, HLB symptoms were not assessable. Plants were pruned after sample collection at 6 mai.

accompanied by reduced leaf size, and blotchy mottle. Symptoms lower in Las-inoculated seedlings (53%) than in mock-inoculated

for the trifoliate hybrids were limited to blotchy mottle, except for seedlings (69%). Increases in stem diameters measured for Benecke,

US-942. The latter displayed a considerable abundance of curled, Carrizo and US-942 at 18 mai were 9%, 40% and 63% on average and

chlorotic and necrotic leaves of much reduced size in 44% of Las- not signiﬁcantly different in mock- and Las-inoculated seedlings.

inoculated plants. The percentage of affected leaves for US-812,

US-942, Cleopatra, and Volkamer at 12 mai varied from plant to 3.3. Experiment 3

plant and ranged from 25% to 75% of leaves. Foliar HLB symptoms on

Carrizo and US-802 were mostly limited to less than 25% of leaves. 3.3.1. Percentage of PCR-positive plants and number of Las

All plants with foliar disease expression were PCR-positive for Las. genomes

At 18 mai, despite the occurrence of some chlorotic leaves, Benecke At 2 mai, none of the US-942 plants were found to be PCR-

seedlings looked healthier compared with 12 mai, and leaves from positive for Las (Table 4). At 4 mai, 15% of Las-inoculated plants

Las-inoculated plants did not look different than leaves from were PCR-positive and the percentage increased to 70% at 12 mai.

mock-inoculated plants. Of the Las-inoculated Carrizo seedlings, The number of Las genomes of PCR-positive plants ranged from

5 6

6 showed blotchy mottling of leaves which was restricted to less 2.7 × 10 to 2.0 × 10 . Las genome numbers for all Las-inoculated

than 25% of leaves. None of the US-942 seedlings displayed any plants were 1.0 × 10 at the end of the experiment.

leaf aberrations at this time. Leaves from mock-inoculated and

Las-inoculated plants of all genotypes are depicted in Fig. 2.

3.3.2. Foliar disease expression

Leaves of Las-inoculated US-942 plants micro-grafted on Volka-

mer lemon did not look different than leaves from mock-inoculated

3.2.3. Growth performance

plants throughout the study and, except for miticide damage at 6

The average shoot mass measured at 12 mai for US-897, US-

mai, no leaf abnormalities were observed.

942, US-812, and Carrizo was between 151 g and 215 g and did

not differ signiﬁcantly (P > 0.05) between mock-inoculated and Las-

inoculated plants (Fig. 3). Lowest shoot mass was observed for 3.3.3. Growth performance

Benecke, and was signiﬁcantly lower in the mock-inoculated con- The average shoot mass of US-942 plants was 69 g at the end

trol (33 g), compared with 17 g in Las-inoculated plants. The aver- of the study (12 mai) and not signiﬁcantly (P > 0.05) different in

age shoot mass of US-802 and Volkamer were considerably higher mock-inoculated and Las-inoculated plants (Table 5). Stem diam-

compared with the other genotypes, but were signiﬁcantly reduced eters increased 23% (scion) and 16% (rootstock) on average during

from 297 g to 246 g in US-802 and from 327 g to 289 g in Volkamer the course of the experiment and no signiﬁcant difference was

in response to inoculation with Las. The average shoot mass of Las- found for mock- and Las-inoculated plants

inoculated Cleopatra seedlings was 137 g and reduced by 31% com-

pared with mock-inoculated plants. Shoot masses for Benecke, Car- 3.4. Experiment 4

rizo and US-942 at 18 mai were 19 g, 54 g and 20 g on average and

did not differ signiﬁcantly (P > 0.05) between mock-inoculated con- 3.4.1. Percentage of PCR-positive plants and number of Las

trols and Las-inoculated PCR-positive plants. Average stem diame- genomes

ter growth measured at 12 mai ranged from 6% in Benecke to 61% in The percentage of PCR-positive US-812 plants ranged from 10%

Cleopatra (Fig. 3). Only stem growth for Cleopatra was signiﬁcantly at 2 mai to 100% at 12 mai (Table 4). The number of Las genomes of

Table 4

Percentage of plants from experiment 3 (US-942) and 4 (US-812) PCR-positive for Ca. L. asiaticus and number of Las genomes 2–12 months after inoculation (mai).

Months after inoculation

2 4 6 9 12

Percentage of PCR-positive plants

US-942 0 15 30 55 70

US-812 10 25 25 95 100

Number of Las genomes (PCR-positive plants)

US-942 – 751,149 711,601 265,475 1,968,045

US-812 1,118,528 2,246,300 203,879 553,615 2,129,310

Number of Las genomes (Las-inoculated plants)

US-942 17 91 668 6309 99,534

US-812 127 754 161 414,614 2,129,310

Numbers of Las genomes are expressed per gram plant tissue. Plants were pruned after sample collection at 6 mai. A total of 20 plants per genotype were analyzed.

76 U. Albrecht, K.D. Bowman / Scientia Horticulturae 147 (2012) 71–80

Fig. 2. Foliar disease expression of plants from experiment 2: (a) Benecke, (b) Carrizo citrange, (c) US-802, (d) US-897, (e) US-812, (f) US-942, (g) Cleopatra mandarin, (h)

Volkamer lemon. Plants in (a) are Las-inoculated (left) and mock-inoculated (right). Leaves in (b–h) are from mock-inoculated plants (left) and Las-inoculated PCR-positive

plants (right). Five cent pieces are depicted for scale.

5 6

PCR-positive plants ranged from 2.0 10 to 2.2 × 10 . Las genome mai. At the end of the study (12 mai), 65% of Las-inoculated plants

numbers for all Las-inoculated plants were 2.1 10 at the end of displayed blotchy mottling of leaves which was not observed in

the experiment. mock-inoculated plants and which was visible on 25–50% of leaves

of affected plants.

3.4.2. Foliar disease expression

Between 2 mai and 9 mai, leaves of Las-inoculated US-812 plants 3.4.3. Growth performance

micro-grafted on Volkamer lemon did not look different than leaves The average shoot mass of US-812 plants was 127 g at the end of

from mock-inoculated plants. However, miticide treatment caused the study and not signiﬁcantly (P > 0.05) different in mock- and Las-

a chlorotic blotching of leaves, hampering leaf evaluation at 4 and 6 inoculated plants (Table 5). Stem diameters increased 32% (scion)

U. Albrecht, K.D. Bowman / Scientia Horticulturae 147 (2012) 71–80 77

400

350 P= 0.0268 P= 0.0164 300

250 P= 0.0025 200 Ctrl Las 150 Shoot mass (g) 100

50 P= 0.0007

0 Benecke Carrizo US-802 US-812 US-897 US-942 Cleopatra Volkamer

80 P= 0.0321

40 Ctrl Las 30

20 Stem diameter growth (%) 10

Benecke Carrizo US-802 US-812 US-897 US-942 Cleopatra Volkamer

Fig. 3. Shoot mass (top) and stem diameter growth (bottom) of mock-inoculated (Ctrl) and Ca. L. asiaticus (Las)-inoculated citrus seedlings from experiment 2 at 12 months

after inoculation. P-values for signiﬁcant (P < 0.05) differences between control- and PCR-positive plants are indicated. Vertical bars are standard errors of the mean. Nine

mock-inoculated and 27 Las-inoculated plants per genotype were analyzed, except for Cleopatra, where 7 and 23 plants, respectively, were analyzed.

and 37% (rootstock) on average throughout the experiment and 2011. The average number of Las genomes was lowest for US-897

no signiﬁcant difference was found for mock- and Las-inoculated throughout the study. In 2011, the highest number measured was

6 6

plants. 8.0 × 10 for US-942 compared with 3.6–4.9 × 10 measured for

US-802 and US-897.

3.5. Field-grown trees

3.5.2. Foliar disease expression

3.5.1. Percentage of PCR-positive plants and number of Las Leaves of all PCR-positive US-802 trees showed the distinct

genomes blotchy mottling typically associated with HLB which, except

The percentage of PCR-positive plants detected in September for 2007, affected the majority of leaves in the canopy. In 2009,

2007 was 5% in US-897 and 16-18% in US-942 and US-802 (Table 6).

In June 2009, all US-802 trees were PCR-positive for Las whereas

Table 6

50% and 68% trees were detected for US-897 and US-942, respec-

Percentage of ﬁeld-grown US-802, US-897 and US-942 trees PCR-positive for Ca. L.

tively. Incidence of detection was 100% for all genotypes in August

asiaticus and number of Ca. L. asiaticus (Las) genomes from 2007 to 2011.

September 2007 June 2009 August 2011

Table 5

Percentage of PCR-positive plants

Shoot mass and scion- and rootstock diameter growth of mock-inoculated (con-

US-802 18 100 100

trol) and Las-inoculated plants (Las) from experiment 3 (US-942) and 4 (US-812) 12

US-897 5 50 100

months after inoculation.

US-942 16 68 100

Number of Las genome (PCR-positive plants)

Control Las P

US-802 n/a 1,697,445 4,861,741

US-942 US-897 n/a 159,332 3,633,964

Shoot mass (g) 74.4 63.8 >0.05 US-942 n/a 3,399,750 7,983,321

Scion diameter growth (%) 20.9 25.0 >0.05 Number of Las genomes (all plants)

Rootstock diameter growth (%) 15.9 16.5 >0.05 US-802 n/a 1,697,445 4,861,741

US-812 US-897 n/a 3816 3,633,964

Shoot mass (g) 139.3 114.0 >0.05 US-942 n/a 143,189 7,983,321

Scion diameter growth (%) 37.0 27.8 >0.05

PCR results are based on conventional PCR according to Albrecht and Bowman

Rootstock diameter growth (%) 41.4 32.2 >0.05 (2008).

Ten (US-942) or nine (US-812) mock-inoculated and 20 Las-inoculated plants per Numbers of Las genomes are expressed per gram plant tissue. A total of 20

genotype were analyzed. (US-802), 19 (US-942) and 17 (US-897) trees per genotype were analyzed.

78 U. Albrecht, K.D. Bowman / Scientia Horticulturae 147 (2012) 71–80

die-back of twigs was observed in all US-802 trees and dead twigs all genotypes, but did not exceed 52% in Benecke and Carrizo 1 year

were often covered with a considerable amount of canker lesions. after inoculation. The percentages for the other genotypes ranged

Canker lesions were also visible on leaves and fruits. In 2011, from 70% to 96% at this time. Although Benecke seedlings had the

US-802 trees were less affected by canker and displayed a fuller lowest number of Las-positive plants, it cannot be ruled out that

canopy compared with the previous years. A faint and difﬁcult to poor growth, independent of inoculation with Las and resulting

discern blotchy mottle was detected in 18% of US-897 trees in 2009 in necrosis and drop of leaves, negatively affected Las detection.

and in 65% of US-897 trees in 2011, which was restricted to a small Poor growth of Benecke seedlings and other cultivars of P. trifoli-

proportion (<10%) of leaves in the canopy. Blotchy mottle was ata is a phenomenon often observed under greenhouse conditions

observed in all US-942 trees in 2009 and 2011, but was limited to (personal observation) and may be associated with different nutri-

10–30% of leaves. US-897 and US-942 trees appeared to be healthy tional requirements and the deciduous nature of this genotype.

and exhibited a full canopy without noticeable twig-die-back Further evaluation of Benecke and Carrizo seedlings 18 months

throughout the study. after inoculation revealed a majority of plants with detectable Las

levels, and Benecke seedlings were much healthier compared with

before. Similar to our ﬁndings, the percentage of graft-inoculated

4. Discussion plants that tested PCR-positive for Las varied in different citrus

species from Malaysia (Shokrollah et al., 2009). The lowest per-

Little resistance to HLB has been reported in citrus and related centage of infected plants was observed for the genotypes Citrus

species, and commercial citrus varieties are particularly susceptible aurantium and Citrus aurantifolia, contrary to C. reticulata for which

to this destructive disease which is found in most citrus-producing all plants tested positive 6 months after inoculation. Lopes and Frare

countries around the world. Among the few varieties reportedly (2008) found different rates of Ca. L. americanus transmission in

unaffected by HLB, are those commonly used as a rootstock, such different citrus genotypes from Brazil, with lowest transmission

as trifoliate orange (McClean and Schwarz, 1970; Miyakawa, 1980) (2%) observed for the trifoliate hybrid rootstock genotype Swingle

and its hybrids Carrizo citrange (Folimonova et al., 2009) and US- citrumelo (P. trifoliata × Citrus paradisi) and highest transmission

897 (Albrecht and Bowman, 2011). This study investigated the (65%) observed for ‘Hamlin’ sweet orange. In contrast, Folimonova

response of several trifoliate hybrid rootstock selections and other et al. (2009) did not ﬁnd obvious differences in the efﬁciencies of

rootstocks to Ca. L. asiaticus (Las), the suspected causal agent of HLB Las transmission to different citrus genotypes in Florida.

in Florida. The dramatic increase in the percentage of infected plants

Graft-inoculation of plants under controlled conditions in the after pruning which was also observed in experiment 1, suggests

greenhouse revealed considerable differences among different that pruning would probably be ineffective to control HLB in the

rootstock varieties tested. In an initial experiment, seedlings of rootstocks studied. The ineffectiveness of pruning, either by remov-

the trifoliate hybrid genotypes US-802 and US-897 were found ing HLB-affected branches or by cutting off the trunk above the

to be little affected by Las compared with Cleopatra mandarin graft-union (decapitation), to control HLB was demonstrated by

seedlings, which displayed strong chlorosis and blotchy mottle Lopes et al. (2007) for ﬁeld-grown sweet orange trees naturally

of leaves and were noticeably stunted. Whereas the majority of infected with Ca. L. americanus in Brazil. Similar to our green-

Cleopatra seedlings were PCR-positive for Las within a few months house studies, the authors found a considerable increase in the

after inoculation, none of the US-802 seedlings had detectable Las percentage of symptomatic trees at the end of their experiments,

levels until nearly a year after inoculation, at which time blotchy 5–9 months after pruning. Contrary to US-897 seedlings, which

mottle was visible in some of the plants. The rate at which Las was remained symptom-free even after prolonged infection, blotchy

detectable in US-897 seedlings was also much reduced compared mottle and chlorosis were detected on leaves of Cleopatra and

with Cleopatra and plants did not display any growth reductions Volkamer seedlings soon after detection of Las by PCR. Trifoli-

or foliar HLB symptoms throughout the study. Las genome values ate hybrids remained symptom-free until 6 months after pruning

were much lower in the trifoliate hybrid genotypes, particularly when blotchy mottle was detected in 26–44% of Carrizo, US-802,

in US-802, throughout the experiment. Susceptibility of Cleopatra and US-812 seedlings. The leaf abnormalities observed for some

and tolerance of US-897 to Las was detected in an earlier study of the infected US-942 plants were only temporary and may have

conducted in our laboratory (Albrecht and Bowman, 2011). It was been part of a defense response, comparable to the hypersensi-

proposed that tolerance may be conferred through antimicrobial tive response observed in other host–pathogen systems. In their

or other compounds associated with the P. trifoliata parentage. The ﬁeld study in Brazil, Lopes et al. (2007) found that the percentage

results from the present study suggest that the trifoliate hybrid of trees with symptoms increased with the severity of symp-

US-802, like US-897, is tolerant to HLB. The higher resistance of toms prior to pruning. In addition to foliar disease symptoms,

US-802 to early infection with Las may be associated with its high signiﬁcant shoot mass reductions were measured in Las-inoculated

vigor (Bowman, 2007a) contrary to US-897, which is a dwarﬁng Benecke-, US-802-, Cleopatra-, and Volkamer-seedlings. Yet, con-

trifoliate hybrid variety (Bowman, 2007b). tinued observation of Benecke revealed no signiﬁcant differences

In the second experiment, response to Las was assessed in eight between control and infected plants at a later time point. The tem-

rootstock varieties, which included the trifoliate hybrids US-802, porary growth reductions of Benecke may have been the result of

US-812, US-897, US-942, and Carrizo citrange, the trifoliate orange additional stress induced by Las in plants already stressed for other

genotype Benecke, as well as Cleopatra mandarin and Volkamer reasons.

lemon. Results of PCR analysis revealed considerable differences A range of HLB disease symptoms was described by McClean

in the rate at which Las was detected in the different genotypes. and Schwarz (1970) for different naturally and graft-inoculated cit-

Consistent with the results from the previous experiment, rates of rus species in South Africa. Similar to the ﬁndings here, trifoliate

detection were highest for Cleopatra where more than half of the orange did not display any well-deﬁned leaf symptoms diagnos-

plants tested PCR-positive between 3 and 6 months after inocula- tic for HLB. Folimonova et al. (2009) analyzed the host response to

tion with Las. In contrast, less than 7% of Benecke, Carrizo citrange, Las in 30 different graft-inoculated citrus cultivars and citrus rela-

US-802, US-942, and Volkamer lemon seedlings were Las-positive tives and observed substantial variations between the genotypes.

during the same time period. Of the inoculated US-812 and US-897 Whereas chlorosis and reduced growth was most common among

seedlings, up to 26% contained detectable Las levels. The percent- the susceptible genotypes, little or no foliar disease symptoms and

age of PCR-positive plants increased considerably after pruning in growth reductions were observed for Carrizo citrange. Results for P.

U. Albrecht, K.D. Bowman / Scientia Horticulturae 147 (2012) 71–80 79

trifoliata seedlings were inconsistent and variable responses were the results presented here which demonstrate reduced rates of

found for Cleopatra mandarin. Lopes and Frare (2008) evaluated infection and little or delayed disease expression in these geno-

the reaction of different Brazilian scion- and rootstock cultivars 15 types. Interestingly, Shokrollah et al. (2011) reported reduced

months after graft-inoculation with Ca. L. americanus and found disease severity in susceptible C. reticulata scion in Malaysia when

that leaf symptoms varied with the cultivar. Typical blotchy mot- using speciﬁc combinations of rootstocks and interstocks. How-

tling was observed in sweet orange, in ‘Murcott’ tangor and to a ever, disease assessment did not extend beyond 6 months after

lesser extent in Cleopatra, while the majority of species showed graft-inoculation.

only yellow leaves similar to those induced by mineral deﬁcien- In conclusion, we propose to classify Carrizo citrange, US-897

cies. Interestingly, chlorosis but no blotchy mottle was observed and US-942 as tolerant, US-812, US-802 and Volkamer lemon as

for the trifoliate hybrid rootstock Swingle citrumelo, despite very moderately tolerant, and Cleopatra mandarin as susceptible to Las.

low transmission efﬁciencies. Similar to the results presented here, Whereas Carrizo, US-802, and Volkamer plants were able to restrict

Lopes and Frare (2008) found Cleopatra mandarin to be the most bacterial multiplication during the early stages of infection but

affected rootstock variety. Whereas these authors used 25 or 50 experienced disease symptoms with increasing bacterial numbers,

plants per genotype, Folimonova et al. (2009) used only 6, which US-897 plants appeared to tolerate Las without being consider-

may explain some of the inconsistencies observed in their study. A ably affected. Although growth irregularities were observed for

study conducted in Malaysia rated C. reticulata and Citrus reshni cv. Benecke, low rates of detectable infection in combination with

Cleopatra among the most susceptible citrus species (Shokrollah low Las numbers measured throughout the experiment are indica-

et al., 2009). In India, Cheema et al. (1982) found that even after tive of some resistance to this pathogen. This is supported by

re-inoculation, bud-inoculated Volkamer lemon plants remained observations on a large number of different P. trifoliata selections

free from infection until 1 year after inoculation. The percentage of located at our USHRL research farm which do not exhibit any dis-

infection in Cleopatra and Carrizo was 80% and 40%, respectively, tinct foliar HLB symptoms or growth abnormalities, contrary to

which is similar to our ﬁndings. However, the number of replicates observations on neighboring trees of other genotypes (unpublished

used in this study was 5 or less and levels of infection were deter- data). Experiments are under way to identify ways to increase

mined by chromatographic analysis of gentisic acid rather than by tolerance of commercially important scion cultivars by utilizing

PCR. tolerance of trifoliate-type rootstock selections. Finally, our stud-

The average number of Las genomes detected in the leaves of ies suggest the importance of using large numbers of replication

inoculated plants was considerably lower in most genotypes com- and repeated analysis of plants over an extended period of time

pared with Cleopatra throughout the study. However, except for when conducting resistance screening to HLB for breeding or other

Benecke and US-897, Las numbers in PCR-positive plants increased purposes.

to levels just 2- to 3-fold below those measured for Cleopatra 1 year

after inoculation. Thus, despite the ability to maintain low bacte-

Acknowledgements

rial levels during the early stages of infection, plants do not seem

to be able to restrict bacterial proliferation for an unlimited period

We thank Lynn Faulkner, Kerry-Shea Worton and Emily Domag-

of time. Higher vigor of plants, particularly US-802 and Volkamer

toy, for excellent technical assistance and Dr. Jude Grosser and Dr.

lemon, may have enabled the plants to contain bacterial numbers

Randy Niedz for suggestions and review of the manuscript. We

more effectively. Folimonova et al. (2009) observed lowest bacte-

are grateful to Dr. Greg McCollum for providing the Las standard

rial titer levels in Carrizo citrange and Severinia buxifolia, whereas

curve and valuable discussions of Ct-values. This research was sup-

most other genotypes exhibited similar titer levels independent of

ported in part by grants from the Florida Citrus Production Research

the severity of the host response.

Advisory Council and the Florida Citrus Research and Development

In additional greenhouse experiments, HLB development was

Foundation. Mention of a trademark, warranty, proprietary prod-

studied for the trifoliate hybrid genotypes US-942 and US-812,

uct, or vendor does not imply an approval to the exclusion of other

grafted onto Volkamer lemon rootstock. Similar to the results from

products or vendors that also may be suitable.

the previous experiments, Las was detected at low rates (0–30%)

in both genotypes during the ﬁrst 6 months after inoculation,

although rates of detection were higher for US-812. The percentage References

of PCR-positive plants increased after pruning, particularly in US-

812 for which distinct blotchy mottle was detected in the majority Achor, D.S., Exteberria, E., Wang, N., Folimonova, S.Y., Chung, K.R., Albrigo, L.G.,

2010. Sequence of anatomical symptom observations in citrus affected with

of plants at the end of the study. No foliar disease symptoms or

huanglongbing disease. Plant Pathol. J. 9, 56–64.

other leaf abnormalities were observed for US-942 and both geno-

Albrecht, U., Bowman, K.D., 2012. Transcriptional response of susceptible and toler-

types did not experience any growth reductions. Observations on ant citrus to infection with Candidatus Liberibacter asiaticus. Plant Sci. 185–186,

118–130.

ﬁeld-grown US-802, US-897 and US-942 trees on different root-

Albrecht, U., Bowman, K.D., 2011. Tolerance of the trifoliate citrus hybrid US-897 (Cit-

stocks detected a superior performance of US-897 trees which

rus reticulata Blanco × Poncirus trifoliata L. Raf.) to Huanglongbing. HortScience

showed little foliar disease symptoms or tree damage despite pro- 46, 16–22.

Albrecht, U., Bowman, K.D., 2008. Gene expression in Citrus sinensis (L). Osbeck fol-

longed pressure from HLB. US-942 trees also did not experience any

lowing infection with the bacterial pathogen Candidatus Liberibacter asiaticus

noticeable damage in response to Las, although bacterial numbers

causing huanglongbing in Florida. Plant Sci. 175, 291–306.

were higher in these plants and blotchy mottle was more abun- Albrecht, U., McCollum, G., Bowman, K.D., 2012. Inﬂuence of rootstock variety on

Huanglongbing disease development in ﬁeld-grown sweet orange (Citrus sinen-

dant. Field-grown US-802 trees expressed severe blotchy mottle

sis [L.] Osbeck) trees. Sci. Hortic. 138, 210–220.

throughout the canopy and exhibited higher Las genome numbers

Bové, J.M., 2006. Huanglongbing: a destructive, newly-emerging, century-old dis-

than US-897 trees during the 4 years of the study. ease of citrus. J. Plant Pathol. 88, 7–37.

Although tolerance of a rootstock does not appear to induce high Bowman, K.D., 2010. Notice to Fruit Growers and Nurserymen Relative to the Naming

and Release of the US-942 Citrus Rootstock. United States Dept. of Agric., Agric.

levels of tolerance to HLB in susceptible scions, it may improve per-

Res. Ser., Beltsville, MD, USA.

formance of infected trees (Nariani, 1981; Albrecht et al., 2012).

Bowman, K.D., 2007a. Notice to Fruit Growers and Nurserymen Relative to the Nam-

Field studies at our laboratory showed that foliar disease symp- ing and Release of the US-802 Citrus Rootstock. United States Dept. of Agric.,

Agric. Res. Ser., Beltsville, MD, USA.

toms of sweet orange scion were reduced in some trees on US-897,

Bowman, K.D., 2007b. Notice to Fruit Growers and Nurserymen Relative to the Nam-

and that trees on US-802 and Volkamer lemon often exhibited less

ing and Release of the US-897 Citrus Rootstock. United States Dept. of Agric.,

canopy damage (Albrecht et al., 2012). This is in accordance with Agric. Res. Ser., Beltsville, MD, USA.

80 U. Albrecht, K.D. Bowman / Scientia Horticulturae 147 (2012) 71–80

Bowman, K.D., 2001. Notice to Fruit Growers and Nurserymen Relative to the Naming Li, W., Hartung, J.S., Levy, L., 2006. Quantitative real-time PCR for detection and

and Release of the US-812 Citrus Rootstock. United States Dept. of Agric., Agric. identiﬁcation of Candidatus Liberibacter species associated with citrus huang-

Res. Ser., Beltsville, MD, USA. longbing. J. Microbiol. Methods 66, 104–115.

Castle, W.S., 2010. A career perspective on citrus rootstocks, their development, and Lopes, S.A., Frare, G.F., 2008. Graft transmission and cultivar reaction of citrus to

commercialization. HortScience 45, 11–15. ‘Candidatus Liberibacter americanus’. Plant Dis. 92, 21–24.

Cheema, S.S., Kapur, S.P., Chohan, J.S., 1982. Evaluation of rough lemon strains and Lopes, S.A., Frare, G.F., Yamamoto, P.T., Ayres, A.J., Barbosa, J.C., 2007. Ineffectiveness

other rootstocks against greening-disease of citrus. Sci. Hortic. 18, 71–75. of pruning to control citrus huanglongbing caused by Candidatus Liberibacter

Davis, M.J., Mondal, S.N., Chen, H., Rogers, M.E., Brlansky, R.H., 2008. Co-cultivation americanus. Eur. J. Plant Pathol. 119, 463–468.

of ‘Candidatus Liberibacter asiaticus’ with actinobacteria from citrus with huan- McClean, A.P.D., Schwarz, R.E., 1970. Greening or blotchy-mottle disease of citrus.

glongbing. Plant Dis. 92, 1547–1550. Phytophylactica 2, 177–194.

Duan, Y., Zhou, L., Hall, D.G., Li, W., Doddapaneni, H., Lin, H., Liu, L., Vahling, C.M., Miyakawa, T., 1980. Experimentally-induced symptoms and host range of citrus

Gabriel, D.W., Williams, K.P., Dickerman, A., Sun, Y., Gottwald, T., 2009. Complete likubin (greening disease). Ann. Phytopathol. Soc. Jpn. 46, 224–230.

genome sequence of citrus huanglongbing bacterium, ‘Candidatus Liberibacter Miyakawa, T., Zhao, X.Y., 1990. Citrus host range of greening disease. In: Aubert,

asiaticus’ obtained through metagenomics. MPMI 22, 1011–1020. B., Tontyaporn, S., Buangsuwon, D. (Eds.), Proc. 4th International Asia Paciﬁc

Edriss, M.H., Burger, D.W., 1984. In vitro propagation of ‘Troyer’ citrange from epi- Conference on Citrus Rehabilitation. FAO-UNDP, pp. 118–121.

cotyl segments. Sci. Hortic. 23, 159–162. Nariani, T.K., 1981. Integrated approach to control citrus greening disease in India.

Fan, J., Chen, C., Yu, Q., Brlansky, R.H., Li, Z.-G., Gmitter Jr., F.G., 2011. Compara- Proc. Int. Soc. Citricult. 1, 471–472.

tive iTRAQ proteome and transcriptome analyses of sweet orange infected by Rosales, R., Burns, J.K., 2011. Phytohormone changes and carbohydrate status in

Candidatus Liberibacter asiaticus. Physiol. Plant. 143, 235–245. sweet orange fruit from huanglongbing-infected trees. J. Plant Growth Regul.

Fan, J., Chen, C., Brlansky, R.H., Gmitter Jr., F.G., Li, Z.-G., 2010. Changes in car- 30, 312–321.

bohydrate metabolism in Citrus sinensis infected with ‘Candidatus Liberibacter Schneider, H., 1968. Anatomy of greening-diseased sweet orange shoots. Phy-

asiaticus’. Plant Pathol. 59, 1037–1043. topathology 58, 262–266.

Folimonova, S.Y., Robertson, C.J., Garnsey, S.M., Gowda, S., Dawson, W.O., 2009. Sechler, A., Schuenzel, E.L., Cooke, P., Donnua, S., Thaveechai, N., Postnikova, E., Stone,

Examination of the responses of different genotypes of citrus to huanglongbing A.L., Schneider, W.L., Damsteegt, V.D., Schaad, N.W., 2009. Cultivation of ‘Candi-

(citrus greening) under different conditions. Phytopathology 99, 1346–1354. datus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ associated

Garnier, M., Danel, N., Bové, J.M., 1984. The greening organism is a gram negative bac- with huanglongbing. Phytopathology 99, 480–486.

terium. In: Garnsey, S.M., Timmer, L.W., Dodds, J.A. (Eds.), Proc. 9th Conference Shokrollah, H., Abdullah, T.L., Sijam, K., Abdullah, S.N.A., 2011. Potential use of

of the International Organization of Citrus Virologists. University of California, selected citrus rootstocks and interstocks against HLB disease in Malaysia. Crop

Riverside, pp. 115–124. Prot. 30, 521–525.

Gottwald, T.R., 2010. Current epidemiological understanding of citrus huanglong- Shokrollah, H., Abdullah, T.L., Sijam, K., Abdullah, S.N.A., Abdullah, N.A.P., 2009. Dif-

bing. Annu. Rev. Phytopathol. 48, 119–139. ferential reaction of citrus species in Malaysia to huanglongbing (HLB) disease

Halbert, S.E.,2005. The discovery of huanglongbing in Florida. In: Proc. 2nd Inter- using grafting method. Am. J. Agric. Biol. Sci. 4, 32–38.

national Citrus Canker and Huanglongbing Research Workshop. Florida Citrus Stuchi, E.S., Girardi, E.A., 2010. Use of horticultural practices in citriculture to survive

Mutual, Orlando, FL, p. H-3. Huanglongbing. Embrapa Mandioca e Fruticultura, Documentos 189, ISSN 1809-

Halbert, S.E., Manjunath, K.L., 2004. Asian citrus psyllids (Sternorrhyncha: Psyllidae) 4996. http://www.infoteca.cnptia.embrapa.br/handle/doc/873095

and greening disease of citrus: a literature review and assessment of risk in Teixeira, D.C., Ayres, A.J., Kitajima, E.W., Tanaka, F.A.O., Danet, J.L., Jagoueix-Eveillard,

Florida. Fla. Entomol. 87, 330–353. S., Saillard, C., Bové, J.M., 2005. First report of a huanglongbing-like disease

Hodges, A.W., Spreen, T.H., 2011. Economic Impacts of Citrus Greening (HLB) in of citrus in Sao Paulo State, Brazil, and association of a new liberibacter

Florida, 2006/07–2010/11. Electronic Data Information Source (EDIS) FE903. species, Candidatus Liberibacter americanus, with the disease. Plant Dis. 89,

University of Florida, Gainesville, FL, http://edis.ifas.uﬂ.edu/fe903 107.

Jagoueix, S., Bové, J.M., Garnier, M., 1994. The phloem-limited bacterium of greening Van Vuuren, S.P., Moll, J.N., 1985. Inﬂuence of the rootstocks on greening fruit symp-

disease of citrus is a member of the alpha subdivision of the Proteobacteria. Int. toms. Citrus Subtrop. Fruit J. 612, 7 and 10.

J. Syst. Bacteriol. 44, 379–386. Zou, X., Li, D., Luo, X., Luo, K., 2008. An improved procedure for Agrobacterium-

Kim, J.S., Sagaram, U.S., Burns, J.K., Li, J.-L., Wang, N., 2009. Response of sweet orange mediated transformation of trifoliate orange (Poncirus trifoliata L. Raf.) via direct

(Citrus sinensis) to ‘Candidatus Liberibacter asiaticus’ infection: microscopy and organogenesis. In Vitro Cell. Dev. Biol. Plant 44, 169–177.

microarray analyses. Phytopathology 99, 50–57.