44 Citrograph July/August 2012

Background The Asian type of huanglongbing

(HLB) is the most destructive and widespread among the three known types of the disease. This type is be-lieved to be heat-tolerant, compared to other types. However, our analysis of scientific and anecdotal evidence sug-gests that summer temperature maxi-ma may play a key role in the disease distribution, spread, and occurrence.

In those regions of the world where HLB is severe, there appears to be com-mon climatic conditions -- year-round warm temperature but with moderate summer temperature maxima.

Recent studies in Florida and Bra-zil confirm that high temperatures in the summer reduce the bacterial popu-lation in the infected plants and in the insect vectors. High summer tempera-tures also significantly reduce the vec-tor’s ability to acquire and transmit the disease.

Lab and field studies are being con-ducted to quantify the impact of high temperature on the bacteria and dis-ease and to develop a heat-based tech-nique for managing the infected groves. The preliminary outcomes are encour-aging; the bacterial titer in the plants was dramatically reduced in all field and lab trials, even eliminated from the infected plants in the best cases.

IntroductionThe Asian type of HLB occurs in

parts of United States, Mexico, Asia, Brazil, and other citrus production areas of the world. The bacterium that causes the disease has long been regarded as heat-tolerant, and so is the insect vector, Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. This type of HLB is associated with the bacterium Candidatus Liberibacter asiaticus (unless further explanation is

Impact of high temperature on huanglongbing for development

of a field management strategyYulu Xia, Xiaoling Deng, Guocheng Fan, Ronald Sequeira, Yu Takeuchi and Ignacio Baez

given, HLB in this article refers to the Asian type).

Despite more than 50 years of re-search on HLB, techniques to manage the disease are limited to the removal of infected plants, control of vector in-sects, and planting pathogen-free seed-lings.

Although removal of infected citrus plants is an epidemiologically sound measure, removal of mature-full-productive trees, and replacing them with younger trees, is economi-cally disruptive for producers. Addi-tionally, there is strong evidence from countries such as China and India that newly planted young trees are more susceptible to the disease than mature trees. Furthermore, newly planted trees are generally more attractive to psyllid adults due to the continuous flushing of young trees, and it takes less time for the bacteria to spread to other parts of a plant, especially into the root system.

Cultural practices, particularly a nutritional-based approach, have been used for disease management since the disease was first reported in Asia more than a century ago. The rationale behind this is straightforward – HLB causes a disruption in plant nutritional transport; foliar application of micro-nutrients can compensate for HLB-induced nutritional deficiencies in the infected plants.

Scientific and anecdotal evidences suggest that the nutritional approach, together with vigorous ACP control

and cultural practices such as sufficient irrigation and fertilization, can prolong citrus production for another five to ten years in the mature plants, depend-ing on species/cultivars. Pomelo (pum-melo) performs best under this type of program, followed by sweet orange, and then mandarin.

Our survey in China indicates that even the most HLB-susceptible man-darin cultivars can maintain a certain level of productivity for five to ten years with sound nutritional and other cultural practices.

However, there is no proof that the nutritional approach can reduce or eliminate the bacteria, reduce the dis-ease severity, or the speed of disease spread. Therefore, it would be highly desirable to develop a complementary technique that can reduce or eliminate bacteria in the infected plants, espe-cially in view of the widely adopted nu-tritional approach in Florida and other HLB-infected regions.

Our data analysis and results sug-gest that a heat-based approach may fill the urgent gap. This brief article highlights some of the major findings. For detail information, readers can re-fer to our peer-reviewed article (Xia et al., 2012).

Evidences of impact of heat and/or summer temperature maximum on HLB

Heat treatment to eliminate the pathogen from scions: Scientists in China/Taiwan and India did exten-sive studies using hot water or other heat-based approaches for producing pathogen-free seedlings. Generally, sci-ons treated with a temperature regime of 45°C to 50°C (113°F to 122°F) for 10 minutes to 1 hour were essentially pathogen-free, judging from symptom expression.

Location Avg. Temperature (ºC)

Raleigh, NC, US 47 (June, 2011)

Immokalee, FL, US 43 (June, 2011)

Fuzhou, Fujian, China 50 (July, 2011)

Yangcu, Guangdong, China 48 (July, 2011)

July/August 2012 Citrograph 45

Since molecular techniques for HLB diagnosis were not available dur-ing this time, the scientific community argued whether the heat treatment eliminated the bacteria or simply sup-pressed the bacterial population tem-porarily. As we will discuss later in this article, based on the latest molecular technology, the bacterial pathogens were likely eliminated or significantly reduced in the cases.

Impact of high summer temperature maximum on the bacterium in plants and in vectors, and disease acquisition and transmission by the insect vector: Surveys and interviews in China re-vealed a wealth of scientific and anec-dotal evidences that high summer tem-perature in certain regions or environ-ments might reduce bacterial titer or even eliminate the bacterium from the infected plants.

Several reports suggest that sum-mer disease transmission using grafting was consistently low. As an example, a four-year study resulted in 7.7 % dis-ease transmission rate in summer vs. 70.8% in other, cooler seasons. It was

observed that the bacterium became undetectable in the infected plants after about three years in the green-houses without the temperature cool-ing system.

Titer is a way of expressing bacteri-um concentration. A higher number means less bacteria, and vice versa.

Recent studies provide us with clear scientific evidence on the impact of high temperature on the disease. A study conducted by Brazilian scien-tists indicated that bacterial titers in infected leaves were reduced dramati-cally once temperature reached 38°C (100.4°F). An exploratory study in South China Agricultural University resulted in significantly reduced bac-terial titer in the infected seedlings by using rain showers to increase leaf sur-face temperatures.

Our study using environmental chambers indicated that bacterial titer in the infected seedlings can be dra-matically reduced, even eliminated, under 4-hour temperature treatments

of 45°C and 48°C (113°F and 118.4°F), respectively.

Evidence from Brazil, U.S., Mex-ico, and China where HLB occurs, in-dicated that bacterial titers were the lowest during the summer, strongly suggesting the negative impact of high temperature on the bacteria. The same correlations between high temperature and low bacterial titer in ACP were also documented recently. Studies in Florida and elsewhere also revealed that bacterial acquisition and disease transmission by ACP were reduced sig-nificantly when temperature reached higher than 30°C (86°F), and there was essentially no disease transmission by ACP when temperature reached high-er than 34°C (93.2°F).

Impact of high temperature on global HLB distribution and occur-rence: Dr. Bové first reported the heat-tolerant nature of Asian type of the disease, in comparison to the African type. Besides the laboratory evidence, the distribution of HLB worldwide ap-parently supports the claim, too. The disease occurs in Saudi Arabia, one of

46 Citrograph July/August 2012

the hottest citrus production areas on the planet. HLB has long been present in other hot areas, such as Punjab, Paki-stan, and Guangdong, China.

However, our analysis of climate data and global HLB occurrence sug-gests that summer temperature may be a key factor limiting HLB distribution and occurrence. We correlated HLB oc-currence with areas having a maximum daily temperature of 38°C (100.4°F) or greater for at least five days in a year.

Our analysis showed that known high incidence HLB regions in the world are not within the areas with high temperature maxima. Although HLB occurs in Saudi Arabia, this area has the “coolest” summer temperature maxima in the region. The same obser-vation applies to Pakistan, another hot citrus production country.

The Punjab province of Pakistan has a long HLB presence; the disease was reported there more than a centu-ry ago, earlier than China. According to scientists who visited Punjab province, ACP populations have been very high. There have been few research activities and/or management practices to ad-

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dress HLB; however, citrus remains a major crop for Pakistan, and it is one of the largest citrus production countries in the world. Few would doubt that the industry in Pakistan should have been long gone. We believe that maximum summer temperature limits disease se-verity and spread here.

One may argue that the less de-structive nature of the disease is not because of summer hot temperatures, but because of the long exposure to the disease and genetic difference and/or diversity in citrus plants in countries such as Pakistan. HLB originated in the Southeast Asia region. Most scientists agree that the family Rutaceae, which citrus plants belong to, also originated in this region. It is reasonable to argue that a long history of disease with plant co-evolution and the genetic diversity in citrus plants in this region may result in less disease severity.

However, so far there is no evi-dence of a true resistant or even tol-erant citrus cultivar in Pakistan, India, and China. In the Chinese province of Guangdong, considered to be ground zero for HLB, citrus plants still suffer

severe disease damage. Chinese scien-tists generally agree that mandarins are more vulnerable to the disease than sweet orange, and pomelo plants are generally regarded as the most “toler-ant” commercial citrus cultivars; how-ever, this observation based on anec-dotal evidence than scientific study.

Spread and distribution of HLB in the U.S. may provide additional evidence on the impact of high tem-perature on the disease. ACP was first reported in Florida in 1998, then the disease in 2005. The disease quickly spread to all citrus production regions of the state. Warm temperatures year-round and lack of extreme high sum-mer temperatures in Florida appear to provide an optimal environment for bacterial multiplication and establish-ment.

In contrast, ACP was first report-ed in Texas in 2001, but the disease was not found until recently (January 2012). It is also interesting to notice that the newly found HLB-infected trees in the Rio Grande Valley of Texas appear in severe condition. This implies that the disease might have been there

July/August 2012 Citrograph 47

Field study using plastic sheeting to raise plant temperature. This photo was taken in China.

for at least three to five years. How-ever, only a few trees in a limited lo-cation are infected. This suggests a low disease spread by ACP in Texas. ACP populations were high in the area. This suggests some other factor(s) may play a role here; we believe it is because of high summer temperatures in the Rio Grande Valley.

Although annual average tempera-tures in the citrus production areas in Florida and Texas are similar, summer temperature maxima in Rio Grande Valley of Texas is much higher than Florida (McAllen,TX: 43°C (109.4°F) vs. Miami, FL: 38°C (100.4°F). The high summer temperature in Texas likely slowed the disease from entering and spread after disease establishment. Southern California citrus production areas share a similar summer temper-ature maxima pattern with southern Texas. We believe that the same story of disease entering and spread will hap-pen there, too.

In summary, summer temperature maxima plays a key role in reducing HLB spread and severity. All severe HLB occurrence areas share a com-

mon climate character – warm tem-perature year-round, but lack of high summer temperature maxima.

Development of heated-based techniques for managing HLB

Small-scale exploratory field stud-ies in using heat-based approaches for controlling HLB were conducted in

China back in the 1960s. Due to poor understanding of temperature dynam-ics in citrus, and lack of reliable tech-niques such as quantitative assessment of bacterial change in the plants and measuring canopy temperature, these studies yielded few results.

Another similar exploratory field study was conducted in South Africa

48 Citrograph July/August 2012

against the African type of HLB. Re-sult indicated that the infected plants covered with a polyethylene tent re-covered from HLB infection, based on symptom expression.

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duct a series of experiments to test our hypothesis. Since our results are being reviewed by fellow scientists, we pro-vide only the highlights in this article.

Understanding temperature dynam-ics in citrus plants in the summer: Using data loggers and thermometers, cano-py temperatures of citrus plants were measured in Florida, North Carolina, Fujian and Guangdong, China. Results indicate that leaf surface (10:00 a.m. to 5:00 p.m.) in the outside of canopy can easily reach temperatures higher than

40°C (104°F) in the summer months (Table 1), leaf temperatures inside of canopy were close to the ambient tem-perature. The result has at least three practical implications.

First, summer temperature in the field should significantly reduce bacte-rial populations, this is why titers are the lowest in the summer discussed above. Second, heat-based treatment in the summer should mainly target the inside canopy, since temperature outside canopy is already high enough to kill the bacterium. Lastly, heat-treat-ment application in late spring and early fall when ambient temperature is optimal for bacterial multiplication might be optimal timing, compared to application in the hot summer.

Laboratory studies of impact of high temperature on bacterium popu-lation: The objectives of these studies were to obtain quantitative data on the effect of high temperature on bacterial titer in the infected plants. This infor-mation is critical to design field studies.

Three studies are being conduct-ed. The infected citrus seedlings were placed in environmental chambers for 4 hours at 45°C to 49°C (113°F to 120.2°F and repeated three times dur-ing an interval of one week. Bacteria titers were measured using qPCR be-fore treatment, three weeks after the last treatment, then every month. Re-sults indicated a statistically significant reduction in bacterial titer in all heat treated plants. In the best case, bacteria were undetectable after treatment.

Two field studies, led by two col-laborators, are being conducted by us-ing plastic sheeting to raise plant tem-perature to 40°C (104°F and higher. Plastic sheeting treatment lasted for 4 hours (10:00 a.m. to 2:00 p.m.) each time, repeated three times. Timing and technique for measuring bacterial ti-ter change before and after treatment were the same as the lab study above. All treated plants had significantly reduced bacterial titers, three weeks and two months (the latest data) after treatment, respectively, compared to the untreated check. The bacteria titers in these plants will be tested on month-ly base until no significant difference is observed between the treated and un-treated check.

The result might be the first field success in demonstrating that bacte-rial concentration can be significantly

July/August 2012 Citrograph 49

reduced by using a heat treatment. Symptom differences between the treated and untreated plants were noted. The newly growing leaves in the treated plants showed no apparent disease symptoms. In contrast, disease symptoms were observed on leaves in the untreated plants.

We believe the results from these ongoing studies provide a proof of concept and critical information for the continuation of future studies. For example, we still have limited informa-tion on the impact of treatment length and timing on the bacteria. We need to collect data on bacterial concentration in roots after heat treatment.

AcknowledgementsThe authors are thankful to many

colleagues in China and the United States for helping us with this study. The authors acknowledge financial resources provided by U.S. Farm Bill funds, China Agriculture Ministry, NC State Univer-sity, USDA-APHIS, Fujian Academy of Agricultural Sciences, and South China Agricultural University.

ReferencesBarbosa, J.C., Eckstein, B., Belasque,

J.J., Bergamin Filho, A., 2011. Preliminary study of comparative acquisition of Can-didatus Liberibacter asiaticus and Ca. L. americanus by Diaphorina citri under dif-ferent temperatures. Proceedings of the 2nd International Research Conference on Huanglongbing, January 10-14, 2011, Orlando, Florida.

Bové, J.M., 2006. Huanglongbing: A destructive, newly emerging, century-old disease of citrus. J. Plant Pathol. 88, 7-37.

Bové, J.M., Calavan, E.C., Capoor, S.P., Cortez, R.E., Schwarz, R.E., 1974. Influence of temperature on symptoms of California stubborn, South African greening, Indian citrus decline and Phil-ippines leaf mottling diseases, in: Weath-ers, L.G., Cohen, M. (Eds.), Proceedings of the 6th Conference of the Interna-tional Organization Citrus Virologists. University of California, Swaziland, pp. 12-15.

Ebert, T.A., Brlansky, R.H., Rogers, M.E., 2011. Seasonal changes in numbers of Asian citrus psyllids carrying Candida-tus Liberibacter asiaticus. Proceedings of the 2nd International Research Confer-ence on Huanglongbing, January 10-14, 2011, Orlando, Florida.

Gmitter, F., Hu, X., 1990. The possible role of Yunnan, China, in the origin of contemporary Citrus species (Rutaceae).

Econ. Bot. 44, 267-277.Gottwald, T.R., Irey, M., Graham, J.,

Wood, B., 2011. Nutritional treatments: Inconsequential effect on HLB control and promote area-wide titer increase and disease spread. Proceedings of the 2nd International Research Conference on Huanglongbing, January 10-14, 2011, Or-lando, Florida.

Halbert, S.E., Manjunath, K.L., 2004. Asian Citrus Psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: a literature review and assessment of risk in Florida. Fla. Entomol. 87, 330-353.

Irey, M., 2011. When should a grower with HLB stop removing trees? , Pro-ceedings of the 2nd International Re-search Conference on Huanglongbing January 10-14, 2011, Orlando, Florida.

Lin, K.H., Lo, H., 1965. A preliminary study on thermotherapy of yellow shoot disease of citrus. Acta Phytophylacica Sin. 4, 169-174.

Lin, K.H., Lo, X.H., Luo, Z.D., 1982b. Heat treatment of citrus seeds for elimi-nating plant pathogens and pests. Acta Phytophylacica Sin. 9, 237-242.

Lopes, S.A., Frare, G.F., Bertolini, E., Cambra, M., Fernandes, N.G., Ayres, A.J., Marin, D.R., Bové, J.M., 2009. Liberibac-ters Associated with Citrus Huanglong-bing in Brazil: ‘Candidatus Liberibacter

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asiaticus’ Is Heat Tolerant, ‘Ca. L. ameri-canus’ Is Heat Sensitive. Plant Disease 93, 257-262.

Morris, R.A., Muraro, R.P., 2011. Economic considerations to treating HLB with the standard protocol or an enhanced foliar nutritional program. Proceedings of the 2nd International Re-search Conference on Huanglongbing January 10-14, 2011, Orlando, Florida.

Nyland, G., Gohhen, A.C., 1969. Heat therapy of virus diseases of perennial

plants. Annu. Rev. Phytopathol. 7, 331-354.

Spann, T.M., Rouse, R.E., Schumann, A.W., 2011. The theory of managing huanglongbing with plant nutrition and real world success in Florida. Proceedings of the 2nd International Research Con-ference on Huanglongbing January 10-14, 2011, Orlando, Florida.

Stansly, P.A., Arevalo, H.A., Rouse, R.E., 2011. Role of nutritional and insec-ticidal treatments in mitigation of HLB: main effects and interactions. Proceed-ings of the 2nd International Research Conference on Huanglongbing, January 10-14, 2011, Orlando, Florida.

Xia, Y., Ouyang, G., Sequeira, R., Chen, J., 2011. A review of managing Huanglongbing (Citrus Greening) us-ing nutritional approach in China. Plant Health Prog.

Xia, Y., Sequeira, R., Takeuchi, Y., Deng, X., Fang, G., Beattie, G., Baez, I., and Yang, R., 2012. Developing a Heat-based Approach for Managing Huan-glongbing (Citrus Greening) Disease in the Field: review and survey results. Crop Protection (Under Review)

Zhao, X.Y., Jian, Y.H., Qiu, Z.S., Su, W.F., Li, C.Y., 1982. A technique of graft transmission of citrus yellow shoot dis-ease. Acta Phytopathol. Sin. 12, 53-56.

Dr. Yulu Xia is Assistant Director, National Science Foundation Center for Integrated Pest Management (CIPM), North Carolina State University, Raleigh, NC. Dr. Yu Takeuchi is a research associ-ate at CIPM. Dr. Ronald Sequeira and Dr. Ignacio Baez are with the USDA-APHIS-PPQ Center for Plant Health Science and Technology, Raleigh, NC. Dr. Xiaoling Deng is with South China Agricultural University, Guangzhou, China. Dr. Guo-cheng Fan is with Fujian Academy of Ag-ricultural Sciences, Fuzhou, China. ●

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