Thursday, April 24, 2008

Xylella fastidiosa

The seminar disclaimer applies to this post.

The topic of seminar this week was: "Xylella fastidiosa: pathogenicity, host specificity, and disease management." The talk was given by Don Hopkins, faculty member of the University of Florida Institute for Agricultural Sciences Mid-Florida Research & Education Center (UF/IFAS-MREC)

Xylella fastidiosa is a bacterium that used to be classified as fastidious, because it was considered to be unculturable. Currently, methods do exist for growing X. fastidiosa.

X. fastidiosa causes economic losses in grape (Pierce's disease), citrus (citrus variegated chlorosis, CVC), almond, coffee, peach, and plum. It is also responsible for decline of many urban shade trees and shrubs. There appear to be 3 subspecies with different host-specificities, which is what part of Dr. Hopkin's research is focused on. He isolated X. fastidiosa from different plant species, and inoculated other plant species with those isolates. He found that isolates were most pathogenic on the plant species they were isolated from, but some isolates could cause disease on several plant species.

The disease
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The bacterium is spread by the glassy-winged sharpshooter, and infects the plant xylem, clogging it, and thereby slowing down the transpiration stream. The vascular obstruction causes symptoms of water stress. Bacterial toxins have also been proposed to be responsible for chlorosis and scorching symptoms, and growth regulators cold be the source of flattened dark green leaves, and shortened internodes which are among the symptoms of infected plants.

Research questions
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One of the questions Dr. Hopkins was trying to answer was whether the virulence of the bacterium is related to the rate of colonization of the xylem vessels, or the movement of the bacterium within the xylem. He found that the ability of X. fastidiosa to colonize the plant systemically as opposed to staying locally at the site of initial infection, determined virulence and host specificity. Since the entire genome of Xylella fastidiosa has been sequenced, researchers can use the hints provided by research results to look for specific genes that are likely involved in pathogenicity and virulence.

Disease management
Because there both the bacterium and the insect vector have a wide host range, and there are a number of plant hosts that show little or no symptoms, it is difficult to sustain efforts to exclude either X. fastidiosa or the sharpshooter. Systemic insecticides, especially within the confines of a vineyard, can to some extent be used to control the insect vector, but is made more difficult by the fact that there are so many plant hosts. Elimination of inoculum sources is another strategy, and involves removal of infected trees.

One approach in the grape industry in California involves application of the soil-applied systemic insecticide Admire in May, monitoring the vineyards, and removal of infected trees.

Plant resistance is of little value in grape culture, where the genotypes of the crop are of immense importance to the quality of the wine. Transgenic resistance does show some promise, and currently there are some field trial underway with grape vines that have be genetically altered to include a lytic peptide gene.

Of major interest is current research that involves the use of a weekly virulent strain of X. fastidiosa obtained from elderberry, which appears to offer cross-protection in the field. Very young plants are inoculated with this strain early on with this mild strain. In one experiment, plants are still healthy more than 10 years after the initial inoculation. Further experiments to test this biological control agent are being conducted in several different states. Interestingly, the treatment is more effective if the initial inoculation is performed with a highly diluted bacterial suspension. The procedure is currently in the patent process. So far there is no data to support the idea that the sharpshooter spreads the biocontrol agent, but this is hard to test, because the protecting strain maintains very low numbers in the plant, and is often hard to detect.

Future research
Current and future experiments focus on the testing of the effectiveness of cross-protection on other grape genotypes, expansion of testing in commercial vineyards in different areas, and the use of X. fastidiosa strains to control other diseases. Data is being collected to submit to the EPA, commercial interest is being evaluated, and experiments are performed to get more data on the efficacy of the treatment.

Further reading
Almeida, R. No year. Xylella fastidiosa-A scientific and community internet resource on plant diseases caused by the bacterium Xylella fastidiosa. Online at:

Mizell, R.F., Andersen, P.C., Tipping, C. and Brodbeck, B. 2008. Xylella fastidiosa diseases and their leafhopper vector. Document ENY-683 (INA174) Department of Entomology and Nematology, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Online at:

Tuesday, April 15, 2008

Liver cancer on citrus

The seminar disclaimer applies to this post.

Seminar was canceled two weeks ago. Last week’s seminar was presented by Mike Irey from the United States Sugar Corporation and Southern Gardens. Southern Gardens is the 3rd largest orange grower in Florida, with 21,000 total grove acreage, all planted with oranges. Over the years more than 21% of the acreage has been lost as a result of citrus canker and the citrus canker eradication effort.

However, if citrus canker was a hemorrhoid, citrus greening (also called huanglongbing, HLB) is the liver cancer of citrus

The disease
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HLB is a bacterial disease, caused by one of the two fastidious (up to now unculturable) bacteria Candidatus Liberibacter asiaticus or Candidatus Liberibacter africanus, the first one of which was found in Florida in 2005. The disease is vectored (spread) by the Asian citrus psyllid, an insect that doesn’t cause any major problems on citrus by itself. When 6 trees in Southern Gardens were identified to have HLB in October 2005, immediate inspection of all groves followed, and a control program to manage the vector, the Asian citrus psyllid was put into place.

Picture credit.
The initial HLB symptoms are misleadingly unimpressive, looking somewhat like nutritional deficiencies. Some leaves, usually near the center of the citrus tree show some mottling, but the trees themselves do not decline until much later. The spread of HLB has been quite dramatic over the years. It was first identified in Florida in 2005. In April 2006, 12 counties had positively identified trees, in January 2007 that was 14 counties, by June of 2007 there were 24 counties, and as of February 2008 all 30 citrus-producing countries had trees with HLB. The disease spreads very fast.

Crop losses include the immediate loss of removed trees, but also gradual tree decline and reduced production, fruit drop, reduced size of the fruit, and possibly juice quality issues, although the latter is still controversial. A small study with 10 pairs of trees comparing yields from symptomatic trees with non-symptomatic trees of approximately the same height, measured a 56% yield reduction in the symptomatic trees. This may be an over-estimate, but it is still a number to keep in mind.

Southern Gardens and US Sugar Corporation test samples from growers by polymerase chain reaction (PCR). They find that around 50-60% of the samples are positive. Some blocks of citrus have 80% of the trees infected, company-wide around 10% of the trees have HLB. Almost 300,000 trees (~1800 acres) have been removed so far, at a cost of more than 6 million dollars (roughly 1 million for tree removal, 2 million for tree replacement, 3 million for lost production). The costs of managing HLB disease has skyrocketed because of the ongoing tree removal, the increased insecticide applications, and currently 44 full-time scouts are employed, constantly surveying the groves in search of newly symptomatic trees.

Grower reactions have varied from doing nothing at all, to aggressive inoculum management, to abandoning groves all together. Those that wait for the silver bullet will likely be disappointed, as there will be no quick fix to the HLB problem.

Disease management
The main challenge is to make management decisions to deal with a disease when there is little knowledge, and a lot of data in the literature is anecdotal. In addition, the causal agent of HLB is a select agent, which complicates research.
Possible management options include:
- Vector control
- Tree removal
- Replanting disease-free material
- Disease-resistance plants (no know resistance to date)

Southern Gardens has opted for an open-door policy, opening their doors for hands-on training of scouts and managers, organizing grower meetings throughout the state, and field trials of experimental approaches.

Management of available inoculum through intensive surveys (full-time scouting) and aggressive removal of infected trees and control of the insect vector, have been the main focus of the approach so far.

The citrus industry has for a long time implemented an integrated pest management approach to control pests and diseases, using cultural practices, surveying, and careful weighing the necessity of chemical sprays. With the current intensive spray program to control the Asian citrus psyllid, the spray programs can no longer be considered IPM.

Management and administration costs have increased dramatically; disease testing for example, costs $6.50 per sample. If the psyllid is found in a nursery, the nursery managers risk have to worry about being assessed and quarantined, risking losses, and potentially even complete shutdown.

Even more troubling is that growers are becoming desperate, trying unproven methods. Often there is no data available from scientifically performed experiments with proper controls on products for which broad claims of efficacy are made. Meanwhile, the growers do not remove infected trees while they try out new products or approaches, risking further spread.

Long term strategies
At this point it is unclear what the best management strategy will turn out to be. One interesting observation to keep in mind in developing strategies is that it appears the disease is more prevalent at the edges of citrus blocks. Potential explanations are that the psyllid is coming from the outside, affecting the outer edges, or the psyllid is moving outward from the center of the block and is stopped by the lack of trees beyond the edge. Does it make sense to change the size and shape of blocks to reduce the edge/area ratio?

How effective are some of the alternative approaches and products that are currently available on the market? How can new strains be detected in a pathogen that is (up to now) unculturable? How does one optimize the PCR reaction used to detect the pathogen (where to set thresholds)? What time of the year is the best to sample? A particular tree may be negative if tested in one month, and positive in another. How effective (and economically feasible) is the strategy to intermingle citrus plants with guava plants, which seems to work in some countries? And can it still be considered a “citrus grove” if half the trees are guava?

Citrus greening is presenting new challenges to growers, nursery managers, plant pathologists, and lab technicians. A great deal of research is necessary to come up with answers.

Further reading
Halbert, Susan. Pest Alert. Florida Department of Agriculture and Consumer Services, Division of Plant Industry.

Langham, M.A.C. 2006. Citrus greening - The yellow dragon threatens Florida citrus. APSnet News and Views.

Long, P. and Merzer, M. 2007. Florida citrus industry faces new peril. Miami Herald. Complete text online at Southern Gardens News and Press Releases:

Wednesday, April 2, 2008

Bacterial fruit blotch of cucurbits

The seminar disclaimer applies to this post.

Seminar this week was given by Dr. Ronald Walcott from the University of Georgia (UGA), Athens. He got his BS and MS degree from Iowa State University, and his PhD from UGA. While searching the internet for references related to this post, I found this interview with Dr. Walcott. Dr. Walcott studies seed-borne plant pathogens.

Picture credit
Bacterial fruit blotch (BFB) has become a significant pathogen over the years, in particular on watermelon. The first symptoms normally appear on the cotyledons, afterwards symptoms can sometimes be seen on the true leaves too. Under field conditions, it is really hard to distinguish these symptoms, because they are rather nondescript. The main problem occurs later in the season, when the fruits show irregularly shaped blotches, the fruits crack, and the insides of the fruit ooze out. Contrary to a NY Times report in May 1994, watermelons are not known to "explode." Still, these are not watermelons you want to buy or eat.

The disease is caused by the bacterial pathogen Acidovorax avenae subsp. citrulli (Aac), formerly considered to belong to the genus Pseudomonas. The disease cycle starts with infected seed, the most important source of primary inoculum. Since watermelons are typically seeded in flats, with many seedlings close together, maintained under high humidity, and watered by overhead irrigation, the pathogen can spread very easily from one seedling to the next, and a single infected seedling can infect the entire tray. The infection of the fruit actually occurs very early on, after the flower opens. The symptoms don't show up until much later. The infected fruit cracks open, seeds come out, can germinate in the soil (volunteer seedlings), and become a source of secondary inoculum.

BFB was first reported in Georgia in 1965, 4 years later it was observed at a research farm in Leesburg, Fl. In 1978 a strain was described that caused seedling blight, and did not show a hypersensitive response (HR) on tobacco or tomato. In 1987 there was an outbreak in the Mariana Islands, and in 1989 a strain was recovered from commercial plantings of watermelons in Florida and Indiana. Contrary to the 1978 strain, this strain caused seedling blight and fruit rot, and elicited an HR on tobacco and tomato. Since 1992, many US states have reported BFB, and there has been a significant amount of yield loss. In addition, growers have filed lawsuits against seed companies, the seed companies have in return restricted watermelon seed sales. Seeds now come with a disclaimer; the growers needs to accept the risk of BFB, and agree not to file any lawsuits.

In 1995, management guidelines were established in Georgia to control the pathogen, and these seemed to limit the problem initially, but then in 1999 and 2000 there were substantial outbreaks. Either the guidelines didn't work, or there was something else going on. Because of the increased movement of seeds, BFB currently occurs worldwide.

In 1999 there were also outbreaks on different crops. BFB was no longer restricted to watermelon, but was observed on canteloupe, melon, and pumpkin (oh no! What does that mean for Halloween?). There were often no obvious symptoms, other than a few small spots on the rind. However, below those spots, the rind doesn't develop, and the fruit goes bad. Some other hosts that the pathogen has been found on include cucumber, honeydew, hami melon, squash, bitter and bottle gourd. Seed transmission has been confirmed for several of these.

Dr. Walcott's lab investigated the genetic diversity of Aac. Using several different techniques, he discovered that at least two groups can be distinguished. Group I strains show less genetic variation than group II strains. Groups I strains have similar, moderate severity on a number of hosts, while group II strains is much more severe on watermelon, while being moderately aggressive on other hosts. The genome of a group II strain has recently been sequences, and annotation of the genome is currently in progress. Dr. Walcott is investigating the possibility of sequencing a group I strain too, so that differences at the genomic level can be correlated with differences in virulence.

Currently, the production of watermelon seed is not really conducive for Aac spread. The seeds are produced under cool, dry conditions, the seed production fields are visually inspected, and the seeds are tested for presence of Aac. But Aac continues to be a problem. Dr. Walcott therefore studied the mechanisms of seed infection. He figured there were 3 possibilities:
1. The bacteria penetrate through the ovary wall. This is unlikely, symptoms would be apparent.
2. The bacteria infect the plant systemically via the vascular system. However, there is no evidence of systemic infection.
3. The bacteria penetrate through the flower parts. Hmmm. This is promising.

Dr. Walcott considered that the bacteria may land on the stigma, move through the style, and end up in the ovary, and thought it the most likely possibility.

He took symptomless fruit, harvested the seed, and found that Aac can associate with seeds, without any symptoms on the fruit. He developed transgenic Aac bacteria expressing green fluorescent protein (GFP), so that he could track the bacteria easily. And indeed, he found the bacteria present in seed tissue, and he could track the bacteria following the path described above. He also noted that this type of infection is not unique to Aac, there are other pathogens that can do this. Using the GFP-tagged bacteria he also found out that the bacteria take about a week to travel from the stigma to the ovary via the pistil pathway. Once the bacteria reached the ovary, however, replication of the bacterium stopped. This explains the lack of symptoms.

Dr. Walcott discovered that bacterial motility was not important for colonization of the seeds, but that pollination was necessary. He also investigated the role of pollinating insects, and found in one experiment that bees did seem capable of spreading the infection, but these results need to be verified with additional experiments. The problem with this theory in practice is, however, that in commercial seed production fields, most pollination does not occur by bees, but by hand, so the bees may not be sufficient to explain the outbreaks seen.

Towards the end of seminar, Dr. Walcott discussed some of the experiments he has done to investigate the possibility of biological control. Although he found a good level of control with at least one biological control agent, this is not good enough for watermelon seed production with a zero-tolerance of Aac.

Further reading

Fessehaie, A., and Walcott, R.R. (2005) Biological control to protect watermelon blossoms and seeds from infection by Acidovorax avenae subsp. citrulli. Phytopathology 95:413-419.

Gitaitis, R.D. and Walcott, R.R. (2007) The epidemiology and management of seedborne bacterial diseases. Ann. Rev. Phytopathology 45: 371-397.

Lessl, J. T., Fessehaie, A. and Walcott, R. R.. 2007. Colonization of female watermelon blossoms by Acidovorax avenae subsp. citrulli and the relationship between blossom inoculum dosage and seed infestation. J. Phytopathology 155:114-121.

Walcott, R.R. (2005) Bacterial fruit blotch of cucurbits. The Plant Health Instructor. DOI:1094/PHI-I-2005-1025-02