The Pressure is on to Eradicate Deadly Organism Affecting Citrus Crops Worldwide

Americans may find a glass of their favorite breakfast staple, orange juice, hard to come by in the next few years if a certain bacteria has its way. According to a spokesperson with the Florida Citrus Mutual, Florida’s orange crop could shrink within five years by about 12%, to 140 million 90-pound boxes, as the state battles the tree-killing disease known as the “Citrus Greening Disease,” or Citrus Huanglongbing.

Citrus Huanglongbing (HLB) – also known as “citrus greening” – is a serious bacterial disease that is adversely affecting citrus groves worldwide. The disease has already been responsible for the significant decrease in citrus production in many countries in Asia, Africa, the Arabian Peninsula, and Brazil. HLB disease, spread by insect called a citrus psyllid, has no known cure , affects all of the main types of citrus, and can ultimately kill the trees. If unstopped, the potential economic impact to the multi-billion dollar citrus industry of the U.S. and the rest of the world could be devastating.

HLB was first detected in Florida in 2005. The Sunshine State’s citrus industry is now mounting a multi-million dollar research effort to try and overcome citrus greening, as the increasing spread of HLB disease is threatening the future of this sector in the U.S.. With commercial citrus production across the entire state now affected by citrus greening, the Florida citrus industry said it plans to carry out extensive research to find short and long-term solutions to the disease.

The 2008-2009 harvests, which ended in July, is forecasted at 159.6 million boxes, down from 170.2 million last year. Growers are awaiting a September tree census to determine how many acres of citrus groves have been lost to citrus greening. The disease was first discovered after a parade of hurricanes battered the groves, and has spread to all thirty-two of Florida’s citrus-growing counties.

At present, the only methods available to curtail citrus greening involve using pesticide to kill the insects, hiring more scouts to inspect the groves, and immediately removing infected trees.

According to Molecular Biologist, Nathan P. Lawrence, Ph.D, Vice President of Marketing at Pressure BioSciences, Inc., researchers have recently sequenced the DNA of the HLB bacterium, an important step toward starting the process of genetically engineering trees to resist the disease. Dr. Lawrence believes a novel process called Pressure Cycling Technology (PCT) may have played a role in helping to extract high quality DNA from the bacterium.

Dr. Lawrence commented, “New sample preparation technologies enable scientists to extract biomolecules related to agriculture and soil-based pathogens more quickly, accurately, and efficiently than ever before. PCT employs cycles of hydrostatic pressure between ambient and ultra-high levels (up to 35,000 psi and greater) to safely, reproducibly, and efficiently release DNA, RNA, and proteins from food, plant, microbial, and other biological samples within minutes, allowing for more rapid and accurate downstream testing.”

Scientists from three separate U.S. Department of Agriculture (USDA) laboratories presented data generated through the use of the Company’s patented, cutting-edge pressure cycling technology (“PCT”) at the American Phytopathological Society’s (“APS”) 2009 Annual Meeting, held August 1-5, 2009, at the Oregon Convention Center in Portland, Oregon. The presentations related to innovative plant pathology studies of various pathogens that can significantly and adversely affect important food crops, such as strawberries, wheat, peas, lentil, barley, canola, and especially citrus.

Dr. Lawrence said, “We are committed to working with plant pathologists in the U.S. and abroad to help improve their understanding of this very serious citrus disease. We are therefore very pleased that PCT combined with our patent-pending ProteoSolve-SB buffer has been reported to extract yields of DNA from cultured HLB that greatly exceed other existing methods.”

Dr. Lawrence continued: “We are also pleased that PCT is being used by a USDA group to extract DNA from fungi in soil that infects wheat and other important crops, and that another USDA group is now evaluating PCT to extract DNA from fungi in soil that infects strawberries. These findings help support the Company’s goal of developing multiple PCT uses in the agricultural area in an effort to help to make PCT the method of choice for the standardization of sample preparation for agricultural applications.”

Know Before You Grow

“Being able to identify a pathogen in existing plants and soil based on its DNA before planting can alert growers to the high risk of failure before they plant, thus helping to avoid failed crops and even potentially financial ruin”, said Dr. Lawrence.

“The problem of contaminated soil and infected plants and trees is not only affecting oranges but other fruits and agricultural products, such as strawberries and wheat – pretty much any staple on any diet on a global scale,” says Dr. Lawrence. “The goal is to thwart global starvation by identifying problem areas, curbing cross contamination, and eradicating the problem before the losses become catastrophic.”

Dr. Norman Schaad (USDA-ARS, Ft. Detrick, MD) presented data on the ability of PCT, when used concomitantly with the ProteoSolve-SB buffer, to extract a yield of double-stranded HLB DNA that was at least 10 times greater than any other DNA extraction method tested. Dr. Schaad also stated that the extracted DNA was of very high quality, and that this 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 a disease and potential disease resistant mechanisms.

Another USDA team, this one including Dr. K.L. Schroeder and colleagues (USDA-ARS, Pullman, WA), reported on the incidence and distribution of Rhizoctonia (causes “damping off”, or the death of seedlings in agriculture) and Pythium (causes “root rot”) fungi species in the soil of wheat, pea, lentil, barley, and canola fields. Fungal DNA was extracted from contaminated soil using PCT, their lab’s standard sample preparation method.

Another team that included Dr. G. J. Bilodeau (USDA-ARS, Salinas, CA) reported on the development of improved tests for the fungus Verticillium dahliae, a pathogen that can cause significant losses in highly susceptible crops such as strawberry. Dr. Bilodeau stated that V. dahliae presents challenges for disruption and extraction of intact DNA, and that he and his colleagues were evaluating different DNA extraction kits, combined with processing by PCT.

Growers are optimistic that the industry can survive if they can control the psyllids, the insect that spreads HLB, and remove infected trees early enough to hang on until resistant trees are available, something that could take a decade. To that end, the recent USDA findings indicate that PCT can be very useful in the laboratory analysis of HLB disease, and consequently may play an important role in the development of new procedures for diagnosing and controlling HLB. “Such advances must be brought to market quickly”, commented Dr. Lawrence, “as we believe that this disease has the potential to significantly adversely affect the citrus industry worldwide.”