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: http://www.cnr.berkeley.edu/xylella/

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: http://edis.ifas.ufl.edu/in174

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.

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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.

Impact
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. http://www.doacs.state.fl.us/pi/chrp/greening/citrusgreeningalert.html

Langham, M.A.C. 2006. Citrus greening - The yellow dragon threatens Florida citrus. APSnet News and Views. http://www.apsnet.org/education/K-12PlantPathways/NewsViews/Archive/2006_04.html

Long, P. and Merzer, M. 2007. Florida citrus industry faces new peril. Miami Herald. Complete text online at Southern Gardens News and Press Releases: http://southerngardens.com/news/101607.html

Late-season vine decline of melons

The seminar disclaimer applies to this post.

This week our departmental seminar was given by none other than the current President of the profession organization for plant pathologists, the American Phytopathological Society (APS), Dr. Raymond D. Martyn. Dr. Martyn is a homegrown graduate, who did his PhD research on biological control of waterhyacinths. He then moved to Texas A&M, where he was a professor for 20 years, and his research focused on soil-borne diseases of melon. He then moved to Purdue University as Head of the Botany and Plant Pathology Department.

Dr. Martyn is no longer involved in active research but this has given him plenty of time to digest the work he did over the years.

During the past 20-25 years a dramatic increase in late-season vine decline (LSVD) of melons has been observed. This involves all members of the cucurbit family of plants, for example watermelon, squash, and muskmelon. LSVD is a generic term used to describe a set of symptoms that include the sudden death of cucurbit foliage about 2-3 weeks before harvest. This period is critical for the accumulation of sugars during the final ripening stages of the melons, so the vine death results in major losses.

There is no single cause for LSVD, nothing that correlates exclusively with vine decline. Sometimes there are pathogens involved, other times not. Vine decline has reportedly been associated with presence of fungi, viruses, bacteria, application of herbicides, and more. The fungus Monosporascus cannonballus, is one of the major fungal pathogens found to be associated with LSVD.

M. cannonballus is a fungus that forms fruiting bodies called perithecia on the roots of the melons. These perithecia have structures in them called asci (singular: ascus). Fungi belonging to the ascomycota typically have asci with 8 ascospores, M. cannonballus is different in that each ascus only has one ascospore, but this spore contains 8 nuclei (it's a called a multinucleate ascospore). Sometimes 16, but mostly 8. Looking at the picture below, it's easy to see where the fungus gets its name from, the perithecium is almost perfectly round and smooth. The ascospores have very thick walls, and are hard to germinate: they are dormant spores. M. cannonballus was first reported in 1970 as a saprophyte (not a pathogen) on canteloupe, in 1983 it was first reported as a plant pathogen in Israel, and in 1989 it was first reported within the US by guess who... Dr. Ray Martyn.

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In the greenhouse, M. cannonballus can infect many different plant species. When a fungus can infect many species, it is said to have a "wide host range." However, M. cannonballus is only a problem on watermelon and muskmelon in the field, so for practical purposes M. cannonballus has a narrow host range. Dr. Martyn studied potential biocontrol methods. He looked at ways in which the disease can be managed using organisms. In this case, M. cannonballus changed in culture, and became hypovirulent, meaning they caused a lot less disease than the original fungus. He investigated whether he could decrease the damage caused in the field by adding the hypovirulent fungus, which would "mate" with the disease-causing fungus, resulting in hypovirulent offspring.

Dr. Martyn authored an article for the APS Education website on Monosporascus root rot and late-season vine decline of melons.

A number of other pathogens were studied as possible causes of late-season vine decline, but all turned out to be fairly weak pathogens, that only caused damage when the plant was very young. In LSVD, the plant is mature, and much stronger, and these pathogens were not responsible for the disease symptoms. In addition, Monosporascus is not the cause of LSVD everywhere in the world. It is reported only in areas that are fairly warm.

Since LSVD is a fairly recent syndrome, Dr. Martyn considered the changes in melon production in the past 25 years. These are:
1. Melon production switched from open-pollinated plots to the use of hybrid melon cultures.
2. Instead of direct seeding into the ground, current production techniques almost exclusively use transplants (seedlings are grown in small trays, and transferred into the ground when they are older).
3. Planting used to be in bare ground, and irrigation systems used furrows, currently plastic mulch and drip irrigation are used.
4. Plants are planted much closer together than in the past.

Dr. Martyn proposed during the seminar that these changes in melon culture practices result in compromised and restricted root systems on the plants. When melons are seeded directly into the ground, the initial root that is formed when a seed germinates, the tap root, continues to grow deep into the soil, as deep as 6-8 feet. A large number of secondary and tertiary roots form along the length of the tap root, allowing the plant to extract water and nutrients from deep in the soil. When seed is germinated in little seedling trays, the tap root grows to meet the bottom of the tray and starts growing in a circle. Once the plant is transplanted into soil, the taproot never recovers, and dies off. Secondary roots that are formed after transplanting do not grow deeply into the soil, but remain about 4" or so below the ground level, and grow essentially horizontally. This drastically limits the availability of water and nutrients to the root system, because the roots do not reach deep enough into the soil.

The compromised root systems have a hard time keeping up with the demand for water of the mature plant, especially during the latter stages, when fruits need to accumulate sugars. Minor pathogens and additional stress, for example lack of water, cause rapid death of the plant in the final stages of fruit development, when the plant needs the resources the most.

Dr. Martyn investigated using cone-tainers, which are very long containers to start seedlings, and they allow for much more growth of the tap root before transplanting, and are so long that the tap root does not reach the bottom before transplanting occurs. After the seedling is planted in soil, the tap root can continue to develop.
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In conclusion, LSVD correlates with cultural changes, resulting in vulnerable root systems that cannot handle multiple stresses.