Combating Citrus Disease

Government, university, and industry researchers are actively investigating ways to combat a disease that has the potential to drastically devastate the citrus industry around the world.

Citrus huanglongbing (HLB), also known as citrus greening disease, has been called the most destructive disease of citrus species in the world. It has already wiped out citrus orchards in China, Brazil, and Thailand and has been found in citrus groves throughout Florida. 

HLB is caused by Gram-negative bacteria of the Candidatus Liberibacter species, which are transmitted by an insect, smaller than a grain of rice, called the Asian citrus psyllid. Symptoms include yellow blotches on the leaves (huanglongbing means yellow dragon in Chinese) and fruit that are small and misshapen and remain green (hence the alternative name citrus greening disease). The disease does not harm humans but affects all types of citrus and ultimately kills the trees, usually within 10 years. There is no known cure.

Studying the Effect on Juice
Elizabeth Baldwin ([email protected]), Research Leader in the Citrus & Subtropical Products Laboratory of the U.S. Dept. of Agriculture’s Agricultural Research Service, Winter Haven, Fla., said that the disease was first discovered in Florida citrus trees in 2005. It’s the most serious problem in the industry, with a serious economic impact, she said, and researchers are looking for resistant varieties and working on genetically engineering resistance. 

Because fruit from infected trees has been reported to have off-flavor and since more than 90% of Florida oranges are processed, Baldwin and her coworkers have been studying the effect of the disease on juice flavor, using trained and consumer panels. They conducted chemical and physical analyses of fruit and juice from healthy and infected orange trees, including symptomatic and asymptomatic fruit from infected trees, to determine the effect on juice and reported their results in the December 23, 2009 online issue of Journal of Agricultural and Food Chemistry (http://pubs.acs.org/doi/abs/10.1021/jf9031958). 

Asymptomatic fruit from infected trees tastes fine, Baldwin said, but symptomatic fruit have flavor problems, including increased bitterness. However, values were generally below reported taste threshold levels, and only symptomatic fruit seemed likely to cause flavor problems. The symptomatic fruit drop off the trees quickly, she added, and don’t generally get into the juice stream. Even if some did get into the processing stream, she said, juice from different varieties is generally blended, so the effect of using symptomatic fruit would be well diluted. 

Charles Sims ([email protected]), Professor in the Dept. of Food Science & Human Nutrition at the University of Florida, Gainesville, and his coworkers have also been looking at flavor changes resulting from the disease, using Hamlin and Valencia oranges from three harvest dates and conducting consumer acceptance tests. 

The quality of the juice from asymptomatic fruit was similar to that of the control, Sims said, but juice from symptomatic fruit was poor in quality, differing in flavor, soluble solids content, and other factors. Consumer acceptance of juice from symptomatic fruit was lower than for the other juices. Although the outside of the symptomatic fruit is green, Sims said, the juice looks about the same as the control juice, and color alone may not be enough to distinguish it. 

The juice from the symptomatic fruit was low in Brix and high in acidity—a pretty bad juice if it gets into the processing stream, Sims said. He added that the symptomatic fruit are quite a bit smaller and stay green and should be easy to sort out in the grading process. The hope is that the affected fruit won’t get into the processing stream even if harvested. But in case some does, the researchers are conducting studies to determine what percentage of affected fruit can be blended with other juice and still be tolerated. 

Russell Rouseff ([email protected]), Professor of Food Chemistry at the University of Florida, Lake Alfred, said that HLB is a weird disease—the tree can be affected but in a very localized manner, branch to branch. The leaves on affected branches are blotchy yellow instead of green, and the fruit on those branches are small and misshapen and remain green. Even though the tree is infected, he said, the fruit from asymptomatic branches have typical color of fruit and leaf, differing very little from uninfected trees. The affected fruit, however, are misshapen and more sour. 

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Rouseff and his coworkers conducted chemical analyses and taste tests of juice from symptomatic and asymptomatic oranges and found that control juice and asymptomatic juices had similar Brix and acid levels but that the symptomatic juice had much lower levels. Limonin concentrations were significantly higher in symptomatic juice than the control juice but still below juice bitterness taste thresholds. Symptomatic juice also had higher levels of terpenes and linalool and lower levels of ethyl butanoate than the control juice and lower levels of esters, aldehydes, and sequiterpenes. There were no consistent differences between asymptomatic and control juices. 

The researchers said that the chemical composition of symptomatic juice resembles that of juice from less-mature fruit. They concluded that the reported off-flavor associated with symptomatic juices probably stems from lower concentrations of sugars and higher concentrations of acid, since all known citrus bitter compounds were either below taste thresholds or absent. 

They reported their results in the December 2009 issue of Journal of Food Science (doi: 10.1111/j.1750-3841.2009.01495.x). 

The simplest solution to maintaining juice quality, Rouseff said, is to keep affected fruit out of the processing stream. Most—90%—of the affected fruit is really small and can be separated out by size and color. The main problem is if it is not sized properly and gets into the processing stream. Then the result is the same type of juice problems that occur when small fruit gets into the processing stream, since there is a lot of peel oil. A little bit is okay, he said, but a lot will give a burn. 

Rouseff said that finding—or even engineering—a cultivar with resistance to HLB would be a wonderful hybridizing opportunity.

Sequencing the Genome
Nathan Lawrence ([email protected]), Vice President of Marketing, Pressure BioSciences Inc., South Easton, Mass., said that researchers are actively working to sequence the DNA of the bacterium that causes HLB, an important step toward starting the process of genetically engineering trees to resist the disease. He said that the pressure cycling technology used in the company’s Barocycler instrument is helping to extract high-quality DNA from the bacterium. The instrument uses cycles of hydrostatic pressure between ambient and ultra-high levels (up to 35,000 psi and greater) to rapidly release DNA and other biological samples. (See “Advances in Sample Preparation” in the December 2009 issue of Food Technology for more information on the technology.) 

Norman Schaad ([email protected]), Research Plant Pathologist, USDA/ARS, Ft. Detrick, Md., has used the instrument to extract at least 10 times more double-stranded HLB DNA than any other DNA extraction method tested, Lawrence said. The extracted DNA was of very high quality and should support the effective sequencing of the genome of the HLB bacterium. Genome sequencing is a very important step in gaining a better understanding of the disease and potential disease-resistance mechanisms, he added, and the recent USDA findings indicate that PCT can be very useful in the laboratory analysis of HLB disease and the development of new procedures for diagnosing and controlling HLB. 

Sequencing of the genes of the bacteria responsible for HLB had previously been hampered by the inability to produce pure cultures of the bacteria. Recently, however, Schaad and his coworkers were successful in developing a culture medium, called Liber A, that produced pure cultures of the three species of the Candidatus Liberibacter bacteria associated with HLB: asiaticus, africanus, and americanus. The pure cultures were shown to produce the disease when inoculated into citrus trees. Since citrus trees have multiple organisms and many kinds of disease, Schaad said, it is important to make sure that the right one is targeted. Schaad and his colleagues isolated a pure culture, inoculated it into the tree, and showed that it causes the disease. This is a fundamental approach in both human and plant pathology, he said. 

The researchers reported their results in the American Phytopathological Society’s journal Phytopathology (http://apsjournals.apsnet.org/doi/abs/10.1094/phyto-99-5-0480). It was the first report of the cultivation and pathogenicity of asiaticus and americanus species associated with HLB, and the society said that it was the most-read paper in Phytopathogy online during 2009. 

Schaad said that he took the cultured cells and used the Barocycler to break them—they are very small and hard, like spores, he said—to extract the DNA, something that has been extremely difficult to do by other means. He said that sequencing of the genome is ongoing, using cultured cells of the asiaticus and americanus species. 

Sequencing of the genome can result in production of unique PCR probes and the ability to learn about the bacteria’s metabolic pathways, growth factors, and enzymes, Schaad said, so that we can develop antibodies against the cells to inoculate plants with the cultured organism. This work can also lead to rapid diagnostic methods and the engineering of resistant varieties.

by Neil H. Mermelstein
a Fellow of IFT, is Editor Emeritus of Food Technology
[email protected]