Journal of Integrative Agriculture 2016, 15(1): 111–119

RESEARCH ARTICLE

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ScienceDirect

Evaluation of thermotherapy against Huanglongbing (citrus greening) in the greenhouse

FAN Guo-cheng1*, XIA Yu-lu2*, LIN Xiong-jie1, HU Han-qing1, WANG Xian-da1, RUAN Chuan-qing3, LU Lian-ming4, LIU Bo3

1 Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, P.R.China2 National Science Foundation Center for Integrated Pest Management, North Carolina State University, Raleigh NC 27606, USA3 Agricultural Bio-Resources Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003, P.R.China4 Zhejiang Citrus Research Institute, Taizhou 318020, P.R.China

AbstractHuanglongbing (HLB, or citrus greening) is the most destructive disease of citrus, which is associated with Candidatus Liberibacter asiaticus (Las). Few management options are available, aside from preventive measures such as removing infected plants, planting disease-free seedlings, and managing the insect vector. In this study, we assessed the efficacy of thermotherapy against HLB under controlled greenhouse conditions. A total of 60 two-year-old, graft-infected Citrus retic-ulata Blanco plants were used. The plants were randomly divided into three groups (45°C, 48°C, and untreated control), with five plants/replicate (rep) and four reps/treatment. The treated plants were placed in phytotrons for a 4-h treatment session, repeated once per week for three consecutive weeks. Disease remission was observed eight weeks post-treat-ment. Real-time PCR assays revealed that Las titers in HLB-affected seedlings were significantly reduced in both 45 and 48°C treatments four weeks after treatment, with the exception of eight plants. In contrast, Las titers in the untreated control plants increased significantly during the same period, with a maximum increase of 28-fold. Except for seven plants, Las titers in the new flushes of treated plants decreased more than 90% eight weeks after treatment. Las titers in mature leaves of treated plants decreased 56 and 60% in average at 45 and 48°C, respectively, eight weeks after treatment. The HLB symptoms and Las titer of seedings were markedly alleviated eight weeks after treatment in both 45 and 48°C treatments. Our results laid a good foundation for the further development of citrus free-disease seedling cultivation and Huanglongbing control in the field. The whole plants were replaced for scion or branch in previous as the research object in this study, and the expression of Huanglongbing symptoms combined with real-time polymerase chain reaction (PCR) were used to evaluate the effect of heat treatment in the greenhouse.

Keywords: Candidatus Liberibacter asiaticus, Citrus reticulata, thermotherapy, bacterial titer

1. Introduction

Huanglongbing (HLB, also known as citrus greening), is the most devastating citrus disease worldwide (Bové 2006). In Asia, the disease is associated with the bacterium Candi-datus Liberibacter asiaticus (Las). Currently, 11 out of the

Received 22 December, 2014 Accepted 6 July, 2015FAN Guo-cheng, E-mail: guochengfan@126.com; Correspondence LIU Bo, Moblie: +86-13905917339, Fax: +86-591-87884262, E-mail: fzliubo@163.com* These authors contributed equally to this study.

© 2016, CAAS. All rights reserved. Published by Elsevier Ltd.doi: 10.1016/S2095-3119(15)61085-1

112 FAN Guo-cheng et al. Journal of Integrative Agriculture 2016, 15(1): 111–119

19 citrus-growing provinces in China suffer serious HLB damage (Fan et al. 2009). Worldwide, the disease is known to occur in more than 40 countries in Asia, Africa, Oceania, South America and North America (Bové 2006). HLB can be spread by Asian citrus psyllid, Diaphorina citri Kuwayama, and African psyllid, Trioza erytreae (McLean and Oberholzer 1965). It can also be transmitted through grafting (Lin 1963).

Presently, there is no commercial citrus cultivar resistant to the disease, few effective measures are available for disease control, although antibiotics such as tetracycline and penicillin are effective against the pathogen (Martinez and Nora 1970; Nariani et al. 1971; Schwarz and Vuuren 1971; GHRG 1976; Bové et al. 1980; Chen et al. 1981; Zhao et al. 1981; Zhao et al. 1982a). Treatment of infected scions and flushes using 0.5–2 g kg–1 tetracycline for 30–120 min resulted in significant disease remission (Ke and Wang 1988). HLB-affected seed-lings of Citrus sinensis (Linn.) Osbeck, Citrus reticulata Blanco cv. Tankan, and C. sinensis Osbeck cv. Huazhou showed disease remission after a treatment of 1 g kg–1 tetracycline hydrochloride solution at 44–50°C (Luo 1991). More recent researches have been conducted using antibiotics and other antimicrobial agents in managing HLB-affected plants (Zhang et al. 2010, 2011). However, several studies indicated that HLB symptoms reappeared less than a year after treatment (Lo et al. 1981). Additionally, effective dosage of antibiotics can be difficult to implement due to variation in the distribution of antibiotics among target plants (Chen et al. 1981). Long-term use of antibiotics may cause Las to develop tolerance or resistance to the agents. Application of antibiotics in citrus may also pose a threat to human health (Smith et al. 2003, 2005; Kelly et al. 2004). A recent study indicated that application of soil conditioner showed promise as another alternative to manage the disease (Xu et al. 2013).

Thermotherapy has been applied in the management of plant diseases for more than 100 years (Zandbergen 1965). For example, the chlorotic leaf spot virus in apple was eradicated using thermotherapy (Campbell 1961; Lister

et al. 1965). Las is generally regarded as a heat-tolerant bacterium (Bové et al. 1974). However, high temperatures can eliminate the bacterium inside scions or seedlings. Lin Kung Hsiang, a Chinese plant pathologist, studied the use of thermotherapy in controlling HLB in the 1960s. His experiments demonstrated the efficacy of hot air (45–51°C) in eliminating HLB-affected scions and seedlings based on symptomatic observations (Lin and Zheng 1964; Lin and Lo 1965). Lo et al. (1981) treated HLB-affected citrus trees using water-saturated hot air at temperature of 49–50°C for 50–60 min, resulted in complete disease remission in all treated seedlings. Hoffman et al. (2013) demonstrated that Las bacteria in HLB-affected seedlings can be completed removed or significantly reduced after a heat treatment under 40–42°C for a minimum of 48 h.

The objective of this study was to demonstrate that a higher temperature schedule with a short 4-h treatment session was effective against HLB. Both quantified change in Las titer before and after treatment and HLB symptom expression were used for evaluating the efficacy of thermo-therapy against HLB-affected plants.

2. Results

2.1. HLB symptom expression change

By the end of eight weeks after heat treatment in the phy-totron, with the temperature 45 or 48°C, new flushes were abundant in the treated plants. The new flushes looked healthy (Fig. 1-B), free of HLB symptoms such as yellow-ing or mottling. All mature leaves on the plants had fallen, except for six plants that retained some mature leaves. Half year after treatment, few typical HLB symptoms were observed in the new flushes. The plants still look healthy. In the case of the untreated control plants, by the end of eight weeks after treatment, most of the mature leaves on the plants had fallen. The remaining leaves showed severe HLB

A B

Fig. 1 Treated plants before and eight weeks after heat treatment in the phytotron, with the temperature 45°C. A, before heat treatment. B, eight weeks after heat treatment.

113FAN Guo-cheng et al. Journal of Integrative Agriculture 2016, 15(1): 111–119

symptoms. A total of six (out of 20) plants had new flushes. Half year after treatment, some of the plants began to die.

2.2. Las titer change - four weeks after treatment

Overall, Las titers in the treated plants decreased signifi-cantly in 45 and 48°C treatments, compared to the untreated control plants. No significant difference in Las titer among the replicates in the treated and the untreated control plants was observed (Table 1). Las titers in three plants showed

a decline of more than 90%, and decreased by more than 50% in 10 (out of 20) plants, with the exception in six plants whose values increased slightly (Table 1) in 45°C treatment. In 48°C treatment, the average decreasing rate of Las titer was 61% except for two plants. The highest decline of Las titer was 90.7%. Eight plants exhibited Las titer reduction of more than 70% (Table 1). Las titers in the untreated plants were significantly increased, except for two plants. Four plants showed Las titer increase of greater than 10-fold, with a maximum of 28-fold (Table 1).

Table 1 Candidatus Liberibacter asiaticus (Las) titer change four weeks after thermotherapy treatment in two-year-old Citrus reticulata Blanco plants in the greenhouse

Biological replicate Tree no.

Before treatment1) After treatment2)

Decreased rate of Las (%)3) Significance4)

Ct0CN0

(copies ng–1) Ct1CN1

(copies ng–1)Untreated control 1st 7 28.10±0.40 2.05E+07 27.34±0.15 3.56E+07 –74.02 a A

17 25.95±0.15 9.78E+07 26.34±0.06 7.37E+07 24.63 9 29.08±0.35 1.00E+07 26.20±0.10 8.16E+07 –711.96

28 28.95±0.42 1.10E+07 24.31±0.20 3.22E+08 –2 815.99 52 27.56±0.21 3.04E+07 24.25±0.16 3.36E+08 –1 008.12

2nd 30 27.32±0.25 3.61E+07 24.12±0.02 3.70E+08 –924.34 a36 27.54±0.35 3.08E+07 23.62±0.28 5.31E+08 –1 626.34 38 28.74±0.51 1.28E+07 24.09±0.02 3.78E+08 –2 843.02 46 25.83±0.23 1.07E+08 26.39±0.35 7.09E+07 33.48 53 25.21±0.16 1.67E+08 24.04±0.40 3.91E+08 –133.57

3rd 49 25.85±0.22 1.05E+08 25.28±0.06 1.59E+08 –51.48 a8 27.15±0.35 4.08E+07 25.04±0.08 1.90E+08 –364.13

48 25.45±0.68 1.40E+08 23.72±0.10 4.95E+08 –252.95 35 26.06±0.25 9.02E+07 24.06±0.12 3.86E+08 –328.04 37 25.55±0.02 1.31E+08 24.77±0.34 2.30E+08 –75.84

4th 42 26.17±0.15 8.34E+07 24.80±0.44 2.25E+08 –170.00 a43 25.07±0.20 1.85E+08 24.04±0.02 3.92E+08 –111.63 51 25.79±0.40 1.10E+08 24.53±0.25 2.74E+08 –150.06 56 23.59±0.21 5.43E+08 21.90±0.30 1.85E+09 –241.17 57 24.02±0.24 3.97E+08 23.38±0.12 6.33E+08 –59.34

45°C treatment 1st 1 24.48±0.35 2.84E+08 25.57±0.06 1.29E+08 54.61 b B4 25.58±0.06 1.28E+08 28.99±0.04 1.08E+07 91.60

13 25.11±0.09 1.80E+08 24.55±0.23 2.70E+08 –50.06 14 24.64±0.08 2.53E+08 23.73±0.20 4.91E+08 –93.54 19 24.12±0.05 3.70E+08 27.80±0.12 2.55E+07 93.10

2nd 22 25.57±0.09 1.29E+08 25.62±0.03 1.24E+08 3.53 b23 26.65±0.08 5.88E+07 27.45±0.05 3.29E+07 44.07 24 25.25±0.06 1.63E+08 26.60±0.08 6.10E+07 62.51 60 25.17±0.40 1.72E+08 23.99±0.19 4.06E+08 –136.05 26 26.09±0.31 8.82E+07 26.94±0.20 4.76E+07 46.03

3rd 27 25.90±0.33 1.01E+08 24.69±0.19 2.44E+08 –141.12 b29 23.03±0.16 8.16E+08 24.22±0.11 3.44E+08 57.86 31 24.14±0.10 3.64E+08 25.38±0.04 1.48E+08 59.36 39 24.48±0.32 2.84E+08 27.81±0.20 2.53E+07 91.09 40 24.74±0.07 2.36E+08 26.02±0.20 9.29E+07 60.58

4th 41 23.79±0.03 4.70E+08 26.19±0.32 8.21E+07 82.55 b47 25.28±0.23 1.59E+08 24.65±0.28 2.51E+08 –57.99 59 24.97±0.27 1.99E+08 25.17±0.13 1.72E+08 13.44 54 24.49±0.55 2.82E+08 24.17±0.16 3.56E+08 –26.53 55 28.10±0.42 2.05E+07 30.43±0.26 3.77E+06 81.58

(Continued on next page)

114 FAN Guo-cheng et al. Journal of Integrative Agriculture 2016, 15(1): 111–119

2.3. Las titer change - eight weeks after treatment

Since new flush leaves appeared eight weeks after treat-ment, PCR assays were conducted separately using both the new and mature leaves. The average Las titer de-creased 90 and 97% in 45 and 48°C treatment, respectively. Las titer declined less than 90% in one plant only in 48°C treatment, and six plants in 45°C treatment (Table 2). Las titers in untreated control plants also decreased. However, the decreasing rates were much less, compared to those in the treated plants. Las titer change differed significantly between treated and untreated control plants (Table 2). In the case of the mature leaves, all mature leaves fell in treated plants, except for six of them. No mature leaf falling was observed in untreated control plants. Las titers were significantly decreased, with an average decrease of 56 and 60% in 45 and 48°C treatment, respectively (Table 3). In contrast, Las titers in the untreated control were significantly increased, with an average increasing of approximate 7-fold. There were significant differences in Las titer in mature leaves between treated and untreated control plants, but no significant difference between 45 and 48°C treatments (Table 3).

3. Discussion

Thermotherapy has been shown to be effective against a wide range of plant pathogens. Positive results have been reported when using hot water or hot air treatments to eliminate bacterial diseases in carrot (Ark and Gardner 1944), tomato (Bakai-Golan 1973; Devash et al. 1980), pea (Grondeau et al. 1992), cowpea (Jindal et al. 1989), barley (Sands et al. 1989), sesamum (Rao and Durgapal 1967), rice (Shekhawat and Srivastava 1971), cabbage (Shekhawat et al. 1982), crucifers (Lin 1981), and bean (Tamietti 1982; Naumann and Karl 1988). Reports also indicated that a single thermotherapy can eradicate several pathogenic organisms (Grondeau et al. 1994).

Results from this study suggest that thermotherapy is a promising option for HLB management. By the end of eight weeks post-treatment, symptom remission was observed, and Las titers declined significantly in the treated plants. Healthy new flushes were abundant in the treated plants. In contrast, HLB symptoms worsened in the untreated control plants four and eight weeks after treatment. Thermotherapy resulted in very low Las titer in new flushes eight weeks after treatment. No symptom remission and Las reduction were

Table 1 (Continued from preceding page)

Biological replicate Tree no.

Before treatment1) After treatment2)

Decreased rate of Las (%)3) Significance4)

Ct0CN0

(copies ng–1) Ct1CN1

(copies ng–1)48°C treatment 1st 2 24.70±0.42 2.42E+08 26.22±0.50 8.02E+07 66.88 c B

3 23.82±0.04 4.60E+08 25.18±0.15 1.71E+08 62.80 10 24.56±0.01 2.69E+08 26.58±0.10 6.19E+07 76.97 11 26.79±0.10 5.31E+07 25.00±0.22 1.95E+08 –266.80 12 24.81±0.05 2.24E+08 26.05±0.51 9.07E+07 59.52

2nd 15 24.68±0.34 2.46E+08 24.02±0.07 3.98E+08 –61.85 c16 25.12±0.10 1.79E+08 25.33±0.11 1.54E+08 14.16 18 25.13±0.06 1.78E+08 28.40±0.07 1.65E+07 90.71 20 25.40±0.10 1.46E+08 26.12±0.02 8.65E+07 40.70 21 25.49±0.12 1.37E+08 27.58±0.02 3.00E+07 78.08

3rd 44 25.30±0.37 1.57E+08 25.82±0.45 1.07E+08 31.50 c25 26.36±0.16 7.26E+07 28.30±0.17 1.77E+07 75.58 32 24.75±0.32 2.34E+08 27.34±0.18 3.56E+07 84.75 33 26.47±0.35 6.69E+07 26.97±0.20 4.66E+07 30.38 34 23.55±0.25 5.59E+08 26.22±0.13 8.04E+07 85.62

4th 42 25.26±0.32 1.61E+08 25.65±0.30 1.21E+08 24.66 c43 24.52±0.35 2.76E+08 25.56±0.12 1.30E+08 52.95 45 24.34±0.27 3.15E+08 26.15±0.14 8.46E+07 73.13 50 24.38±0.40 3.05E+08 26.50±0.25 6.55E+07 78.54 58 26.03±0.17 9.23E+07 27.68±0.04 2.79E+07 69.82

1) Ct0, Ct value (means±standard deviation) in three individual tests before treatment; CN0, Las titer before treatment, according to Ct value (means). The same as below.

2) Ct1, Ct value (means±standard deviation) in three individual tests after treatment; CN1, Las titer after treatment, according to Ct value (means).

3) Decreased rates of Las were calculated using the formula: (CN0–CN1)/CN0×100%. Negative values mean higher Las population.4) Uppercase letters indicate statistically significant differences between the three treatments (i.e., 45°C vs. 48°C vs. untreated control),

and lowercase italic letters indicate statistically significant differences between the four replicates in a treatment at P<0.05, determined by Data Processing System (DPS) using Duncan’s new multiple range method. The same as below.

115FAN Guo-cheng et al. Journal of Integrative Agriculture 2016, 15(1): 111–119

Table 2 Ca. Liberibacter asiaticus titer change in the new flushes eight weeks after thermotherapy treatment in two-year-old C. reticulata Blanco plants in the greenhouse

Biological replicate Tree no.

Before treatment After treatment1)

Decreased rate of Las (%)2) Significance

Ct0CN0

(copies ng–1) Ct2CN2

(copies ng–1)Untreated control3) 7 28.10±0.40 2.05E+07 30.48±0.21 3.64E+06 82.23 a B

46 25.83±0.23 1.07E+08 26.81±0.25 5.23E+07 50.94 53 25.21±0.16 1.67E+08 25.92±0.32 9.98E+07 40.37 48 25.45±0.68 1.40E+08 27.05±0.16 4.40E+07 68.61 56 23.59±0.21 5.43E+08 24.42±0.35 2.97E+08 45.34 57 24.02±0.24 3.97E+08 24.35±0.28 3.12E+08 21.34

45°C treatment 1st 1 24.48±0.35 2.84E+08 27.44±0.02 3.32E+07 88.33 A4 25.58±0.06 1.28E+08 30.60±0.21 3.33E+06 97.40

13 25.11±0.09 1.80E+08 30.74±0.18 3.01E+06 98.33 14 24.64±0.08 2.53E+08 30.58±0.15 3.38E+06 98.66 19 24.12±0.05 3.70E+08 30.70±0.11 3.10E+06 99.16

2nd 22 25.57±0.09 1.29E+08 26.79±0.32 5.31E+07 58.85 a23 26.65±0.08 5.88E+07 27.22±0.02 3.89E+07 33.88 24 25.25±0.06 1.63E+08 30.73±0.15 3.03E+06 98.14 60 25.17±0.40 1.72E+08 30.65±0.17 3.22E+06 98.13 26 26.09±0.31 8.82E+07 30.55±0.18 3.46E+06 96.08

3rd 27 25.90±0.33 1.01E+08 27.90±0.06 2.37E+07 76.57 a29 23.03±0.16 8.16E+08 30.32±0.26 4.08E+06 99.50 31 24.14±0.10 3.64E+08 30.48±0.16 3.64E+06 99.00 39 24.48±0.32 2.84E+08 30.64±0.21 3.24E+06 98.86 40 24.74±0.07 2.36E+08 30.34±0.08 4.03E+06 98.29

4th 41 23.79±0.03 4.70E+08 25.76±0.30 1.12E+08 76.15 a47 25.28±0.23 1.59E+08 30.47±0.23 3.66E+06 97.70 59 24.97±0.27 1.99E+08 30.36±0.17 3.97E+06 98.01 54 24.49±0.55 2.82E+08 29.02±0.24 1.05E+07 96.27 55 28.10±0.42 2.05E+07 30.66±0.26 3.19E+06 84.41

48°C treatment 1st 2 24.70±0.42 2.42E+08 29.87±0.31 5.66E+06 97.66 b A3 23.82±0.04 4.60E+08 30.36±0.21 3.97E+06 99.14

10 24.56±0.01 2.69E+08 30.49±0.27 3.61E+06 98.66 11 26.79±0.10 5.31E+07 30.55±0.22 3.46E+06 93.50 12 24.81±0.05 2.24E+08 30.70±0.14 3.10E+06 98.62

2nd 15 24.68±0.34 2.46E+08 30.46±0.28 3.69E+06 98.50 b16 25.12±0.10 1.79E+08 30.54±0.19 3.48E+06 98.05 18 25.13±0.06 1.78E+08 29.90±0.10 5.55E+06 96.88 20 25.40±0.10 1.46E+08 30.35±0.23 4.00E+06 97.26 21 25.49±0.12 1.37E+08 30.49±0.13 3.61E+06 97.36

3rd 44 25.30±0.37 1.57E+08 30.67±0.17 3.17E+06 97.98 b25 26.36±0.16 7.26E+07 30.62±0.22 3.29E+06 95.47 32 24.75±0.32 2.34E+08 30.34±0.24 4.03E+06 98.28 33 26.47±0.35 6.69E+07 30.65±0.15 3.22E+06 95.20 34 23.55±0.25 5.59E+08 30.17±0.32 4.55E+06 99.19

4th 42 25.26±0.32 1.61E+08 27.85±0.26 2.46E+07 84.76 b43 24.52±0.35 2.76E+08 30.68±0.21 3.15E+06 98.86 45 24.34±0.27 3.15E+08 30.52±0.24 3.53E+06 98.88 50 24.38±0.40 3.05E+08 29.17±0.33 9.41E+06 96.92 58 26.03±0.17 9.23E+07 30.41±0.23 3.83E+06 95.85

1) Ct2, Ct value (means±standard deviation) in three individual tests after treatment, according to Ct value (means); CN2, Las titer after treatment, according to Ct value (means).

2) Decreased rates of Las were calculated using the formula: (CN0–CN2)/CN0×100%3) Few new flushes appeared.

116 FAN Guo-cheng et al. Journal of Integrative Agriculture 2016, 15(1): 111–119

observed in the untreated control plants. Hoffman et al. (2013) observed no HLB symptom on the new flushing after a 10-day thermotherapy session. Las titer had a 989-fold decline 90 days after treatment at a temperature as low as 40°C. The fact of significant Las titer reduction after thermotherapy in this study suggests that Las bacteria in the canopy were largely killed by thermotherapy, since the PCR assays were conducted at least four weeks after last treatment. Josephson et al. (1993) proved that bacterial genomic DNA can remain stable for up to three weeks after cell death. It is unlikely that the bacteria could have migrated to other parts of plants, such as trunk or the root system because these parts were subjected to the same tempera-ture treatments in this study. No significant difference in Las titer was observed between 45 and 48°C treatments four and eight weeks after treatment. This observation suggests

that a temperature of 45°C might be high enough to cause Las mortality in citrus. However, since the bacteria can’t be cultivated, we can’t verify the result by running lab test. We also observed stable Las titer after treatment in few treated plants in both 45 and 48°C treatments. We can’t explain this inconsistence in Las titer dynamic after thermotherapy, a further study is needed.

Although this study demonstrated the efficacy of thermo-therapy for HLB management, critical issues remain unan-swered with regard to the potential for field application. For example, the optimal treatment duration under 45 or 48°C condition remains undetermined. Although Las titers were dramatically reduced, even undetectable, in most treated plants during the study period, it is unclear how long these treated plants can maintain low Las titer or pathogen-free. It is reasonable to speculate that the remaining Las would

Table 3 Ca. Liberibacter asiaticus titer change in the mature leaves eight weeks after thermotherapy treatment in two-year-old C. reticulata Blanco plants in the greenhouse

Biological replicate Tree no.

Before treatment After treatment1)

Decreased rate of Las (%)2) Significance

Ct0CN0

(copies ng–1) Ct3CN3

(copies ng–1)Untreated control 1st 7 28.10±0.40 2.05E+07 25.02±0.15 1.92E+08 –838.95 a A

17 25.95±0.15 9.78E+07 26.67±0.23 5.79E+07 40.77 9 29.08±0.35 1.00E+07 26.34±0.25 7.36E+07 –632.63

28 28.95±0.42 1.10E+07 24.87±0.15 2.15E+08 –1 844.09 52 27.56±0.21 3.04E+07 23.81±0.31 4.62E+08 –1 423.87

2nd 30 27.32±0.25 3.61E+07 25.01±0.42 1.93E+08 –435.00 a36 27.54±0.35 3.08E+07 23.24±0.19 7.00E+08 –2 176.74 38 28.74±0.51 1.28E+07 23.93±0.35 4.24E+08 –3 197.90 46 25.83±0.23 1.07E+08 22.79±0.21 9.71E+08 –810.53 53 25.21±0.16 1.67E+08 25.46±0.33 1.39E+08 16.71

3rd 49 25.85±0.22 1.05E+08 23.42±0.28 6.14E+08 –484.21 a8 27.15±0.35 4.08E+07 23.50±0.24 5.80E+08 –1 319.24

48 25.45±0.68 1.40E+08 22.36±0.48 1.32E+09 –845.45 35 26.06±0.25 9.02E+07 23.90±0.32 4.33E+08 –380.11 37 25.55±0.02 1.31E+08 28.02±0.24 2.17E+07 83.41

4th 42 26.17±0.15 8.34E+07 25.25±0.20 1.63E+08 –95.05 a43 25.07±0.20 1.85E+08 23.37±0.35 6.37E+08 –243.51 51 25.79±0.40 1.10E+08 24.71±0.16 2.41E+08 –119.55 56 23.59±0.21 5.43E+08 23.32±0.26 6.61E+08 –21.63 57 24.02±0.24 3.97E+08 23.89±0.22 4.37E+08 –9.92

45°C treatment3) 13 25.11±0.09 1.80E+08 25.82±0.33 1.07E+08 40.40 A14 24.64±0.08 2.53E+08 25.83±0.10 1.07E+08 57.88 26 26.09±0.31 8.82E+07 27.38±0.02 3.46E+07 60.75 31 24.14±0.10 3.64E+08 25.62±0.15 1.24E+08 65.89 47 25.28±0.23 1.59E+08 25.72±0.32 1.15E+08 27.41 59 24.97±0.27 1.99E+08 27.30±0.04 3.67E+07 81.57

48°C treatment3)

　

10 24.56±0.01 2.69E+08 25.96±0.06 9.72E+07 63.85 A15 24.68±0.34 2.46E+08 26.24±0.15 7.92E+07 67.76 18 25.13±0.06 1.78E+08 28.42±0.29 1.62E+07 90.86 32 24.75±0.32 2.34E+08 27.60±0.07 2.95E+07 87.37 33 26.47±0.35 6.69E+07 25.97±0.07 9.65E+07 –44.10 50 24.38±0.40 3.05E+08 28.76±0.06 1.27E+07 95.84

1) Ct3, Ct value (means±standard deviation) in three individual tests after treatment; CN3, Las titer after treatment, according to Ct value (means).

2) Decreased rates of Las were calculated using the formula: (CN0–CN3)/CN0×100%. Negative values mean higher Las population.3) Most mature leaves fell in 45 and 48°C treatments after treatment.

117FAN Guo-cheng et al. Journal of Integrative Agriculture 2016, 15(1): 111–119

recover to reproduce, and the titer would at some point reach pathogenic levels. Previous experience using antibiotics for HLB management might provide further insights. After treatment with antibiotics such as tetracycline, HLB-infected plants remained symptom-free for about one year (Gao and Zheng 1981; Lo et al. 1981; Ke and Wang 1988). Repeated applications of antibiotics were necessary to maintain grove productivity.

Thermotherapy can be regarded as a “green” technol-ogy, repeated applications may not cause human health or environmental concerns. However, thermotherapy might be associated with additional economic burdens to growers, especially in the developing world. Another concern regarding thermotherapy for HLB management is the Las in the root system. Las titers in roots can be significant once the infection becomes systemic (Li et al. 2009). At present, the efficacy of thermotherapy on Las in roots, either under laboratory conditions or in the field, remains unknown. Laboratory studies are underway to further explore this topic.

4. Conclusion

The HLB symptoms and Las titer of seedings were markedly alleviated eight weeks after treatment in both 45 and 48°C treatments. The new flushes were abundant and looked healthy. Las titers in the new flushes and in mature leaves of treated plants were decreased more than 90 and 56% eight weeks after treatment, respectively. This laboratory study demonstrates that thermotherapy can significantly reduce Las titers in the canopy of HLB-infected plants. Further laboratory and field work are still necessary to address the questions raised above.

5. Materials and methods

5.1. Experimental plants

Two-year-old citrus plants, with Poncirus trifoliate as root-stock and C. reticulata Blanco as scions, were used in this study. Three to four HLB-symptomatic scions were grafted to each plant (Zhao et al. 1982b). The grafted plants were then placed in a screened greenhouse, with conventional irrigation and fertilization schedules. Severe HLB symptoms appeared about six months after grafting. These plants were then subjected to PCR confirmation of HLB presence. Briefly, HLB-symptomatic leaves were collected and washed with tap water. After air drying, total DNA was extracted from leaf midrib tissue (approximately 400 mg) using the cetyltri-ethylammonium bromide (CATB) extraction method (Murray and Thompson 1980) and subjected to nested PCR (Zhang et al. 2009) and real-time PCR (Li et al. 2006). Las-positive

plants were used in the experiments.

5.2. Thermotherapy procedure

Sixty Las-positive plants were randomly divided into three groups: 1) 45°C treatment, 2) 48°C treatment, and 3) un-treated control. Each treatment contained four replicates, with five plants per replicate. Phytotrons (PRX-450D, Saife Instruments, Ningbo) were used to apply thermotherapy. Five HLB-affected plants were placed in a phytotron for a 4-h session of treatment, which was repeated once per week for three consecutive weeks. Relative humidity inside the phytotrons was kept at 95%. The treated plants were then maintained in a screened greenhouse at temperatures of 25–32°C. Routine fertilization and irrigation schedules were implemented after the treatments.

5.3. Sample collection and DNA extraction

Leaf samples were collected from the treated and untreated control plants four and eight weeks after the last thermother-apy (3rd treatment). According to Josephson et al. (1993), bacterial genomic DNA can remain stable for up to three weeks after cell death. Because new flushes (young leaves) were abundant eight weeks after treatment, both mature and young leaves were collected for PCR assay. Leaf samples were washed with tap water and air dried. Leaf midribs (approximately 400 mg) were obtained, cut into segments of 1–2 mm, and ground with a mill (MM400, Retsch GmbH, Germany) after freeze drying. Total DNA was extracted using the CTAB approach described above. The yield and purity of DNA samples were estimated by measuring OD260 nm/280 nm (optical density) and OD260 nm/230 nm, respectively, with a spectrophotometer (NanoDrop 2000c, Thermo Sci-entific, USA). The DNA was used as a template for PCR assays after the concentrations were adjusted to 50 ng μL–1 and stored at –28°C (Li et al. 2007).

5.4. PCR assays and quantification of Las

The primers and probe sequences, based on Li et al. (2006), were synthesized by TaKaRa Biotechnology (Dalian) Co., Ltd. (China). The real-time PCR amplifications were per-formed using a CFX96TM (Bio-Rad, USA) in a 20-μL reaction volume consisting of the following reagents at optimized con-centrations: 10 μL Premix Ex TaqTM, 1 μL 250 nmol L–1 target primer (HLBas and HLBr), 1 μL 150 nmol L–1 target probe (HLBp), 1 μL sample DNA and 6 μL sterile double-distilled water. The standard amplification protocol was 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 58°C for 40 s. All reactions were performed in triplicate, and each run contained one negative and one positive control.

118 FAN Guo-cheng et al. Journal of Integrative Agriculture 2016, 15(1): 111–119

5.5. Statistical analysis

To evaluate the changes of Las titers before and after ther-motherapy in the citrus seedlings, statistical analysis using ANOVA, Duncan’s multiple range test was completed with Data Processing System (DPS) ver. 7.05. All real-time PCR Ct results displayed the detecting level of Las 16S rDNA. A standard equation curve (Y=–3.1692X+19.31) was devel-oped based on the primer and probe designed by Li et al. (2006). The new equation was used to quantify bacterial populations as cells per microgram of total DNA. The num-ber of Las copies was calculated based on molecular weight using the formula: Number of copies=(Amount in nano-grams×Avogadro’s number)/(Length in base pairs×109×650) (Tatineni et al. 2008). The average weight of a base pair is assumed to be 650 daltons and Avogadro’s number is 6.022×1023. For example, 50 ng of pMD 18-T (2 692 bp) consists of approximately 1.72×1010 plasmids.

Acknowledgements

This research was funded by the United States Department of Agriculture (USDA)-APHIS-PPQ-CPHST and North Carolina State University joint project (2012-0195-01) and the Special Fund for Agro-Scientific Research in the Public Interest, China (201003067-05). Thanks to Dr. Ronald Se-queira (USDA-APHIS, USA) for providing technical advice on this study.

References

Ark P A, Gardner M W. 1944. Carrot bacterial blight as it affects the roots. Phytopathology, 34, 415–420.

Bakai-Golan R. 1973. Postharvest heat treatment to control Alternaria tenuis Auct. rot in tomato. Phytopathologia Mediterranea, 12, 108–111.

Bové J M. 2006. Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology, 88, 7–37.

Bové J M, Bonnet P, Garnier M, Aubert B. 1980. Penicillin and tetracycline treatments of greening disease-affected citrus plants in the glasshouse, and the bacterial nature of the procaryote associated with greening. In: Calavan E C, Garnsey S M, Timmer L W, eds., Proceedings of the 8th Conference of the International Organization of Citrus Virologists. University of California, Riverside, USA. pp. 97–102.

Bové J M, Calavan E C, Capoor S P, Cortez R E, Schwarz R E, Weathers L G, Cohen M. 1974. Influence of temperature on symptom of California stubborn, South African greening, Indian citrus decline and Philippines leaf mottling disease. In: Weathers L G, Cohen M, eds., Proceedings of the 6th Conference of the International Organization of Citrus

Virologists. University of California, Berkeley, USA. pp. 12–15.

Campbell A I. 1961. The effect of rubbery wood virus on the stoolbed production of two clonal apple rootstocks. Journal of Horticultural Science, 36, 268–273.

Chen N R, Dai Y M, Wu T X, Xu T. 1981. Control of yellow shoot disease of citrus by trunk injection of tetracycline hydrochloride and some other medicines. Acta Phytophylacica Sinica, 8, 163–169. (in Chinese)

Devash Y, Okon Y, Henis Y. 1980. Survival of Pseudomonas tomato in soil and seeds. Phytopathology, 99, 175–185.

Fan G C, Liu B, Wu R J, Li T, Cai Z J, Ke C. 2009. Thirty years of research on citrus huanglongbing in China. Fujian Journal of Agricultural Sciences, 24, 183–190. (in Chinese)

Gao R X, Zheng H M. 1981. On control of yellow shoot disease of citrus. Acta Phytophylacica Sinica, 8, 53–58. (in Chinese)

Grondeau C, Ladonne F, Fourmond A, Poutier F, Samson R. 1992. Attempt to eradicate Pseudomonas syringae pv. pisi from pea seeds with heat treatments. Seed Science and Technology, 20, 515–525.

Grondeau C, Samson R, Sands D C. 1994. A review of thermotherapy to free plant materials from pathogens, especially seeds from bacteria. Critical Reviews in Plant Sciences, 13, 57–75.

GHRG (Guangxi Huanglongbing Research Group). 1976. The pathogen of huanglongbing and the study on using acheomycin and terramycin to control huanglongbing. Citrus Scientific and Technical News Report, 2, 28–31. (in Chinese)

Hoffman M T, Doud M S, Williams L, Zhang M Q, Ding F, Stover E, Duan Y P. 2013. Heat treatment eliminates ‘Candidatus Liberibacter asiaticus’ from infected citrus trees under controlled conditions. Phytopathology, 103, 15–22.

Jindal K K, Thind B S, Soni P S. 1989. Physical and chemical agents for the control of Xanthomonas campestris pv. vignicola from cowpea seeds. Seed Science and Technology, 17, 371–382.

Josephson K L, Gerba C P, Pepper I L. 1993. Polymerase chain reaction detection of nonviable bacterial pathogens. Applied and Environmental Microbiology, 59, 3513–3515.

Ke C, Wang Z S. 1988. Study on the use of antibiotics to control citrus huanglongbing. Fujian Journal of Agricultural Sciences, 3, 1–10. (in Chinese)

Kelly L, Smith D L, Snary E L, Johnson J A, Harris A D, Wooldridge M, Morris J G. 2004. Animal growth promoters: To ban or not to ban? A risk assessment approach. International Journal of Antimicrobial Agents, 24, 205–212.

Li W B, Hartung J S, Levy L. 2007. Evaluation of DNA amplification methods for improved detection of ‘Candidatus Liberibacter species’ associated with citrus huanglongbing. Plant Disease, 91, 51–58.

Li W B, Levy L, Hartung J S. 2009. Quantitative distribution of ‘Candidatus Liberibacter asiaticus’ in citrus plants with citrus huanglongbing. Phytopathology, 99, 139–144.

Li W B, Hartung J S, Levy L. 2006. Quantitative real-time PCR for detection and identification of Candidatus Liberibacter

119FAN Guo-cheng et al. Journal of Integrative Agriculture 2016, 15(1): 111–119

species associated with citrus huanglongbing. Journal of Microbiological Methods, 66, 104–115.

Lin C Y. 1981. Studies on black rot of cruciferous crops caused by Xanthomonas campestris pv. campestris in Taiwan. Plant Protection Bulletin (Taiwan), 23, 157–167.

Lin K H. 1963. Further studies on citrus yellow shoot. Acta Phytophylacica Sinica, 2, 243–251. (in Chinese)

Lin K H, Lo H H. 1965. A preliminary study on thermotherapy of yellow shoot disease of citrus. Acta Phytophylacica Sinica, 4, 169–174. (in Chinese)

Lin K H, Zheng R Y. 1964. A preliminary study on citrus yellow shoot disease virus and resistance of the diseased citrus budwood to heat. Acta Phytopathologica Sinica, 7, 61–65. (in Chinese)

Lister R M, Bancroft J B, Nadakavukaren M J. 1965. Some sap-transmissible viruses from apple. Phytopathology, 55, 859–870.

Lo X H, Luo Z D, Tang W W. 1981. Studies on the thermotherapy of citrus yellow shoot disease. Acta Phytophylacica Sinica, 8, 47–52. (in Chinese)

Luo Z D. 1991. Preliminary report on the efficacy of heated tetracycline hydrochloride solution in treating citrus yellow shoot disease. Journal of South China Agricultural University, 12, 38–42. (in Chinese)

Martinez A L, Nora D M, Armedilla A L. 1970. Suppression of symptoms of citrus greening disease in the Philippines by treatment with tetracycline antiobiotics. Plant Disease Reproter, 54, 1007–1009.

McClean A P D, Oberholzer P C J. 1965. Citrus psylla, a vector of the greening disease of sweet orange. South African Journal of Agricultural Science, 8, 297–298.

Murray M G, Thompson W F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8, 4321–4326.

Nariani T K, Raychaudhuri S P, Viswanath S M. 1971. Response of greening pathogen of citrus to certain tetracycline antibiotics. Current Science, 40, 552.

Naumann K, Karl H. 1988. Possibilities of disinfecting bean seeds infected with Pseudomonas syringae pv. phaseolicola. Nachrichtenblatt fur den Pflanzenschutz in der DDR, 42, 204–208. (in German)

Rao Y P, Durgapal J C. 1966. Seed transmission of bacterial blight disease of Sesamum (Sesamum orientale L.) and eradication of seed infection. Indian Phytopathology, 19, 402–403.

Sands D C, Fourest E, Rehms L D. 1989. Dry heat seed treatment for Xanthomonas campestris pv. translucens. In: Klement Z, ed., 7th International Conference on Plant Pathogenic Bacteria. June 11–16, 1989. Budapest AkademiaiKiado, Budapest. p. 51.

Schwarz R E, Van Vuuren S P V. 1971. Decrease in fruit greening of sweet orange by trunk injection of tetracyclines. Plant Disease Reporter, 55, 747–750.

Shekhawat G S, Srivastava D N. 1971. Control of bacterial leaf-

streak of rice (Oryza sativa L.). Indian Journal of Agricultural Sciences, 41, 1098–1101.

Shekhawat P S, Jain M L, Chkravarti B P. 1982. Detection and seed transmission of Xanthomonas campestris pv. campestris causing black rot of cabbage and cauliflower and its control by seed treatment. Indian Phytopathology, 35, 442–447.

Smith D L, Dushoff J, Morris J G. 2005. Agricultural antibiotics and human health. PLoS Medicine, 2, 0731–0735.

Smith D L, Johnson J A, Harris A D, Furuno J P, Perencevich E N, Morris J G. 2003. Assessing risks for a pre-emergent pathogen: Virginiamycin use and the emergence of streptogramin resistance in Enterococcus faecium. The Lancet Infectious Diseases, 3, 241–249.

Tamietti G. 1982. Seed treatment trials to control halo blight of bean. Informatore Fitopatologico, 32, 47–50. (in Italian)

Tatineni S, Sagaram U S, Gowda S, Robertson C J, Dawson W O, Iwanami T, Wang N. 2008. In planta distribution of ‘Candidatus Liberibacter asiaticus’ as revealed by polymerase chain reaction (PCR) and real-time PCR. Phytopathology, 98, 592–599.

Xu M R, Liang M D, Chen J C, Xia Y L, Zheng Z, Zhu Q, Deng X L. 2013. Preliminary research on soil conditioner mediated citrus Huanglongbing mitigation in the field in Guangdong, China. European Journal of Plant Pathology, 137, 283–293.

Zandbergen M. 1965. Hot water treatment for bulbs. In: Royal Horticultural Society Daffodil-Tulip Yearbook. London the Royal Horticultural Society, UK. p. 187.

Zhang M Q, Duan Y P, Powell C A. 2010. Bactericidal activities of antimicrobial molecules against huanglongbing-associated ‘Candidatus Liberibacter asiaticus’ in the diseased periwinkle. Phytopathology, 100, S145-S145.

Zhang M Q, Duan Y P, Zhou L J, Turechek W W, Stover E, Powell C A. 2009. Screening molecules for control of citrus huanglongbing using an optimized regeneration system for ‘Candidatus Liberibacter asiaticus’ infected periwinkle (Catharanthus roseus) cuttings. Phytopathology, 100, 239–245.

Zhang M Q, Powell C A, Zhou L J, He Z L, Stover E, Duan Y P. 2011. Chemical compounds effective against the citrus huanglongbing bacterium ‘Candidatus Liberibacter asiaticus’ in planta. Phytopathology, 101, 1097–1103.

Zhao X Y, Jiang Y H, Qiu Z S, Su W F, Li C Y. 1982a. A technique of graft transmission of citrus yellow shoot disease (Huanglongbing). Acta Phytopathologica Sinica, 12, 53–55. (in Chinese)

Zhao X Y, Qiu Z S, Su W F, Jiang Y H. 1981. The reaction of penicillin and tetracycline on citrus Huanglongbing. South China Fruits, 4, 17. (in Chinese)

Zhao X Y, Qiu Z S, Su W F, Jiang Y H. 1982b. Continued effect of tetracycline on the elimination of citrus yellow shoot by budwood immersion. Acta Phytophylacica Sinica, 9, 67–68. (in Chinese)

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