An Emerging Disease Caused by Pseudomonas syringae pv. phaseolicola Threatens Mung Bean Production in China
Suli Sun, Ye Zhi, and Zhendong Zhu, National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, China; Jing Jin, Agronomy and Plant Protection College, Qingdao Agricultural University, Qingdao, 266109, China; and Canxing Duan, Xiaofei Wu, and Wang Xiaoming, National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences
An emerging bacterial disease with symptoms resembling those of halo blight is threatening mung bean production in China. This study was conducted to investigate the disease’s geographic distribution in China using consecutive multiyear field surveys and to confirm the causative agents’ identity. The surveys were conducted in 15 provinces covering seven geographic regions from 2009 to 2014. The survey results revealed that the emerging mung bean disease has rapidly spread and is prevalent in three of the main Chinese geographic regions, which contain more than 90% of the mung-bean-growing areas in China. To confirm the causal agent, diseased mung bean leaves were collected from the surveyed fields and used to isolate the pathogen. A bacterium was consistently isolated from all of the collected leaves. Based on the phenotypic characteristics, the physiological and biochemical properties, pathogenicity tests, and fatty acid composition, in combination with specific polymerase chain reactions and 16S-23S ribosomal DNA sequence analyses, the bacterium was identified as Pseudomonas syringae pv. phaseolicola. To our knowledge, this is the first report of P. syringae pv. phaseolicola causing halo blight on mung bean in China. The results indicate that P. syringae pv. phaseolicola is likely of epidemiological significance on mung bean in China.
Plants have specialized immune receptor proteins on the surface of their cells, which detect specific molecular patterns, or ligands, on harmful bacteria. New research now reveals that these immune receptors, along with the ligand that activates them, must be taken up inside the plant cell in order to mount a full immune response to bacterial infection.
Pseudomonas syringae at stomata opening. This is a confocal microscopy image of Arabidopsis leaf surface infected with Pseudomonas syringae pv tomato DC3000 bacteria. These bacteria, fluorescing green, are present at the stomatal pores in order to invade the leaf. The chloroplasts are highlighted in purple.
Credit: Michael Kopischke, The Sainsbury Laboratory
Plants have specialised immune receptor proteins on the surface of their cells, which detect specific molecular patterns, or ligands, on harmful bacteria. New research by scientists at The Sainsbury Laboratory in Norwich now reveals that these immune receptors, along with the ligand that activates them, must be taken up inside the plant cell in order to mount a full immune response to bacterial infection.
When plants are attacked by harmful organisms, running away clearly isn’t an option — but that doesn’t mean they are powerless. Plants have clever molecular mechanisms that help them to detect and resist pests and pathogens. Scientists at The Sainsbury Laboratory in Norwich are interested in understanding how these immune processes work because this may be useful in breeding disease-resistant crops — especially when climate change threatens to bring new pests and diseases our way.
Working with an international team of collaborators, a new study, led by The Sainsbury Laboratory’s Professor Silke Robatzek and published in the journal Proceedings of the National Academy of Science of the USA, reveals new details about plant-triggered immunity.
Professor Robatzek explained, “We already knew that special protein molecules called ‘pattern recognition receptors’, the immune receptors located on the surface of plant cell membranes, can recognise specific ‘microbe-associated molecular patterns’, or MAMPs, from harmful bacteria. An immune receptor called FLS2 is a well-studied example — this can detect flagellin, a protein found in the tube-like filaments emanating from some bacterial cells that allows them to move. Several other receptors and the associated MAMPs or danger signals that activate them have been discovered, so we know that plants can detect and resist many different types of bacteria, but we were interested in what happens next — when the receptor is activated, what does it do? Where does it go? And how, exactly, does this help the plant to defend itself?”
To answer these questions, the research team used a technique called live cell imaging in which receptor proteins of interest were tagged with a fluorescent marker so that their movements could be visualised using a special microscope.
From previous work, the scientists knew that when FLS2 is activated, it is ‘internalised’. This means that it is moved from the plant cell membrane to a membrane-bound ‘bubble’ inside the cell called an endosome.
Postdoctoral scientist and co-first author of the study Dr Gildas Bourdais, said, “We discovered that several other receptors follow the same pattern as FLS2 — they too are internalised into endosomes, and they require another protein called clathrin to do so. We now show this is a common process conserved among many different immune receptors, and even danger-sensing receptors. Furthermore, we think that the process of internalisation is key to recycling the receptors so that plants stay in ‘defence mode’ in the long run.”
Plants may not be able to run away, but they do have several chemical weapons at their disposal, and can also ‘batten down the hatches’ by closing their stomata — the leaf pores that usually open to allow gas exchange, but which can be infiltrated by harmful bacteria. The scientists observed that stomata closed only when the receptor internalisation mechanism was functional, strongly suggesting that it is key to providing a level of immunity even before bacteria enter the plant.
Dr Bourdais said, “Having lots of specialised immune receptors means that plants can sense many different MAMPs, either from the same bacterial pathogen or different ones — but what happens next — clathrin-mediated internalisation of activated receptors — is a critical step for the plant to fully deploy it’s immune responses to pathogen attack.”
Malick Mbengue, Gildas Bourdais, Fabio Gervasi, Martina Beck, Ji Zhou, Thomas Spallek, Sebastian Bartels, Thomas Boller, Takashi Ueda, Hannah Kuhn, and Silke Robatzek. Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases. Proceedings of the National Academy of Sciences, September 2016 DOI: 10.1073/pnas.1606004113
Bacterial speck disease creates unattractive black spots on a ripe tomato, making it unmarketable.
Researchers at the Cornell-affiliated Boyce Thompson Institute (BTI) and Virginia Tech have discovered a new weapon in the arms race between plants and pathogenic bacteria, which tomatoes use to detect the microbe that causes bacterial speck disease.
The team identified a new receptor in tomato plants, called FLAGELLIN-SENSING 3 (FLS3), which triggers defenses against a bacterial attack. FLS3 is present in a small number of plant species, including tomato, potato and pepper. The study appears in Nature Plants.
“This is an interesting example of a receptor that appears to have evolved fairly recently because it is only found in a small group of plants,” said first author Sarah Hind, a research associate in the lab of BTI professor Gregory Martin. “This discovery sets up the possibility of introducing FLS3 into other economically important crop plants, which might provide resistance to bacterial pathogens that is not naturally present in those plants.”
FLS3 detects a part of the flagellum, a tail-like appendage that helps bacteria swim through their environment and consists mostly of flagellin proteins. When bacteria invade the plant, the FLS3 receptor binds to a region of the flagellin protein called flgII-28 and triggers an immune response.
FLS3 is the second flagellin sensor discovered in tomatoes. The first, called FLS2, is found in most land plants and detects invading bacteria through recognition of a separate region of flagellin. Several bacterial species have acquired mutations that change the shape of their flagellin so that FLS2 can no longer recognize it. The acquisition of the FLS3 receptor may, therefore, serve as a countermeasure on behalf of the tomato to detect bacteria with altered flagellin.
“Discovering that some heirloom tomatoes, such as Yellow Pear, did not respond to flgII-28 was key to using a genetics approach to identify FLS3,” said senior author Martin, the Boyce Schulze Downey Professor at BTI and a Cornell professor in the School of Integrative Plant Science.
In the current paper, Hind used Yellow Pear tomatoes together with a wild relative of tomato called Solanum pimpinellifolium to identify the FLS3 gene and show how it functions to reduce bacterial growth. But to confirm that FLS3 is the receptor for flgII-28, she needed to demonstrate the two molecules can physically interact. Researchers in the laboratories of Martin and Frank Schroeder, BTI associate professor, developed biochemistry techniques to identify a stable complex between FLS3 and flgII-28, thus validating FLS3 as the flgII-28 receptor.
“Proving direct interactions of biomolecules has remained a huge challenge, and our work will help in developing better approaches for exploring receptor-ligand interactions,” said co-author Schroeder, who is also a Cornell professor in chemistry and chemical biology.
The study demonstrates how versatile the plant immune system can be while fighting a constant battle against infectious bacteria. “Plants are always coming up with new ways to defeat pathogens,” said Hind. “We’re trying to understand how they do it and then use this knowledge to develop more disease-resistant plants.”
Additional BTI researchers on the project include research associate Susan Strickler and graduate students Joshua Baccile and Jason Hoki. Former BTI researchers include postdoctoral scientists Patrick Boyle and Zhilong Bao, research assistant Diane Dunham, graduate student Inish O’Doherty, and undergraduate intern Elise Viox.
Patricia Waldron is the staff science writer for the Boyce Thompson Institute.
Courtesy of University of Wisconsin A potato plant shows symptoms of dickeya, a bacterial pathogen long found in Europe that is now spreading in the United States.
Courtesy of Amy Charkowski University of Wisconsin plant pathologist Amy Charkowski.
PARK CITY, Utah — A plant pathologist has advised the potato industry to prioritize research and testing to combat a new threat to U.S. spud production — the bacterial pathogen Dickeya dianthicola.
University of Wisconsin associate professor Amy Charkowski, who also directs Wisconsin’s seed potato certification program, told growers at the National Potato Council’s summer meeting dickeya caused heavy damage to spud fields throughout the East Coast in 2015 and has been troublesome again this season.
Charkowski said the pathogen has posed a major challenge to European potato production since the 1950s, but it didn’t surface in the U.S. until the fall of 2014, when a sample from the Northeast tested positive. It’s since been confirmed in most of the major potato states, including Idaho, North Dakota, Texas, New Mexico, Indiana, Ohio, Wisconsin, Pennsylvania, North Carolina, New York, New Jersey, Maryland, Maine and Florida. It’s also been found in Canada in New Brunswick and Ontario.
“If it gets too entrenched in the seed system, it could be a real problem,” Charkowski said. “I’m really worried about seed testing right now. We don’t have the capacity to test in our system.”
Charkowski said the bacteria can survive in irrigation water and thrives in warm, humid environments and poorly ventilated potato cellars. She said dickeya is easily confused with its close relative, pectobacterium, which is common in states including Idaho and causes similar “black leg” symptoms, including curling and wilting leaves, low emergence and stem-base rot.
Charkowski said dickeya symptoms may remain latent, and it takes fewer dickeya bacteria to infect a plant. Both pathogens need an open wound to infect tissue.
No resistant commercial varieties have been developed. Fortunately, European researchers say the pathogen doesn’t tend to survive longer than nine months in soil, and it can be effectively controlled by good sanitation practices, Charkowski said.
Charkowski said a researcher from Scotland has been working with Maine growers to understand dickeya, and she and several colleagues applied for a grant to work with him as a consultant. She’d also like funding to research environmental conditions that favor dickeya and how to keep dickeya from spreading on seed cutters, believing seed is the primary way the disease spreads. She would also like to see seed certification programs single out black leg in test results.
University of Idaho Extension potato storage specialist Nora Olsen spotted symptoms of dickeya at the Kimberly Research and Extension Center in 2015, after learning of them during a meeting with Maine growers. The center’s samples tested positive, along with about six other samples subsequently submitted that season by commercial growers, she said. Charkowski said her lab tested a positive sample from an Idaho commercial potato farm this season.
Idaho Potato Commissioner Ritchey Toevs, of Aberdeen, advocates dickeya testing of nuclear and first-generation seed as part of his state’s seed certification program, using samples growers must already submit for ring rot testing. Though Toevs acknowledges Idaho’s conditions appear to be unfavorable for widespread dickeya, he noted, “We see problems come up so quickly, and you don’t know which ones are big threats and which ones you can live with.”
Washington Potato Commission Executive Director Chris Voigt said dickeya is on his state’s radar, though it hasn’t been confirmed in Washington, and he sees no need to commit resources toward it, yet.
Disease has spread widely across U.S. citrus region
South Texas citrus threatened by HLB disease.
The war against Citrus Greening disease goes back to 2005 when Florida’s lucrative citrus industry was assaulted by Asian Citrus psyllids, which led to the devastation of much of the state’s citrus crop and created concern among U.S. citrus growers nationwide at the prospect of facing the threat.
That war against the psylid and the plant-killing bacteria it carries (huanglongbing, better known as HLB) has spread over the last 10-plus years as the pests migrated from the Southeast coast westward to Texas, finally reaching California’s and the rich citrus growing areas.
While advances in research on both the citrus disease and the psyllids that vector it from state to state and country to country have intensified through the years, new technology and defense systems have been and are continuing to be developed in agriculture’s ongoing battle against citrus greening.
A new tool being tested may add to the arsenal of weapons to manage and control the spread of huanglongbing by targeting the reproductive activity of the insect that spreads the disease.
HLB is one of the most destructive diseases of citrus worldwide. Originally thought to be caused by a virus, it is now known to be caused by unculturable phloem-limited bacteria. Three forms of greening have been described so far. The African form produces symptoms only under cool conditions and is transmitted by the African citrus psyllid Trioza erytreae; the Asian form prefers warmer conditions and is transmitted by the Asian citrus psyllid Diaphorina citri. Recently, a third American form transmitted by the Asian citrus psyllid was discovered in Brazil. This American form of the disease apparently originated in China.
The findings offer hope of limiting the impact of Xylella fastidiosa that experts described as one of the “most dangerous plant pathogens worldwide”.
If it is not controlled, it could decimate the EU olive oil industry.
The study, carried out by Italian researchers and funded by the European Food Safety Agency (EFSA), began in 2014 and consisted of two main types of experiment: artificial inoculation (via needle) and inoculation via infected vectors (insects) collected from the field.
The tests were carried out on a variety of species, including a range of olive, grape, stone-fruit (almond and cherry) and oak varieties.
“The first results are coming from the artificial inoculation because the field experiments began in the summer so it is only six months old, therefore only part of the results are available,” Giuseppe Stancanelli, head of the EFSA’s Plant and Animal Health Unit, told BBC News.
“The key results are that, 12-14 months after artificial inoculation on different olive varieties, the team found that young plants typically grown in the region displayed symptoms of the dieback.
“The research team also found evidence of the bacterium moving through the tree – towards it root system as well as towards the branches.”
But he added: “What has also been shown is that some varieties have shown some tolerance. They grow in infected orchards but do not show strong symptoms, as seen in more susceptible varieties.
“They are still infected by the inoculation but this infection is much slower so it takes longer for the infection to spread, and the concentration of the bacterium in the plant is much lower.
Dr Stancanelli added that these results were important in terms of providing information for tree breeders.
However, it was too early to say whether or not the olive yields from the varieties that have displayed tolerance to the infection are nonetheless reduced or adversely affected, he observed.
The EFSA Panel on Plant Health produced a report in January warning that the disease was known to affect other commercially important crops, including citrus, grapevines and stone-fruit.
However, the results from the latest experiments offered a glimmer of hope.
“Olives seemed to be the main host of this strain while citrus and grapes did not show infection, either in the field or by artificial inoculation,” Dr Stancanelli said.
He added that the infection did not spread through the citrus and grape plants that were artificially inoculated, and the bacterium was not found beyond the point it was introduced to the plant by injection.
But he added that more research was needed on stone-fruit species.
“The tests on the artificially inoculated varieties of stone-fruit need to be repeated because there is a mechanism in the plants that makes artificial inoculation difficult,” Dr Stancanelli explained.
“Another uncertainty we had was about (holm) oak. Quercus ilex is a typical Mediterranean oak that grows in the landscape and is natural vegetation.
“At the beginning of the outbreak in 2014, some symptoms were found on oaks and the tests were positive but this was never confirmed so this was probably a ‘false positive’.
“The artificial inoculation test appears to have shown that the holm oak is resistant (to the disease).”
The Xylella fastidiosa bacterium invades the vessels that a plant uses to transport water and nutrients, causing it to display symptoms such as scorching and wilting of its foliage, eventually followed by the death of the plant.
Since it was first detected in olive trees in Puglia, southern Italy, in October 2013, it has been recorded in a number of other locations, including southern France. To date, it has yet to be recorded in Spain, the world’s largest olive oil producer.
Experts warn that should the disease, which has numerous hosts and vectors, spread more widely then it has the potential to devastate the EU olive harvest.
Globally, the EU is the largest producer and consumer of olive oil. According to the European Commission, the 28-nation bloc produces 73% and consumes 66% of the the world’s olive oil.
Recent reports suggest that the X. fastidiosa outbreak has led to a 20% increase in olive oil prices during 2015.
In November 2015, the European Commission announced it was providing seven million euros (£5m) from the EU Horizon 2020 programme to fund research into the pathogen.
One of the areas of the Horizon 2020-funded research will be on plant selection to strengthen tolerance and resistance to the disease.
Dr Stancanelli explained that the experiments established in this study would continue as part of the EU-funded Ponte programme.
“The experimental field realized within the pilot project will serve as unique source of plant material for future project actions aiming at investigating the host-pathogen interactions,” he said.
“Investigations will be extended to an additional panel of 20 cultivars which will be planted in… April in the same plot.”
The disease plagued citrus farmers in North and South America for decades. It remained confined on these continents until the mid-1990s when it was recorded on pear trees in Taiwan.
According to the European and Mediterranean Plant Protection Organisation (EPPO), which co-ordinates plant protection efforts in the region, the pathogen had been detected prior to 2013 by member nations on imported coffee plants from South America. However, these plants were controlled and the bacterium did not make it into the wider environment.
Earlier this year, when a cold-tolerant subspecies of the bacterium was identified in the southern France outbreaks, UK government plant health officials published information for horticulture professionals, especially those importing plants. They were advised of their obligations – such as obtaining the necessary plant passports – and given details of the visible symptoms to look out for on potentially infected plants.
Current methods to control the spread of citrus greening are limited to aggressive psyllid control and infected trees removal.
The Asian citrus psyllid carries citrus greening bacterium in its saliva and infects trees when feeding.Tyler Jones, UF/IFAS.
Although current methods to control the spread of citrus greening are limited to aggressive psyllid control and the removal and destruction of infected trees, researchers are working to defeat it on a number of fronts, including suppressing the psyllid, breeding citrus rootstock that shows better greening resistance and testing chemical treatments that could be used on trees.
International researchers, including ones at the University of Florida and Florida State University, are sharing in a $4 million grant from the United States Department of Agriculture to attack the problem of citrus greening, a disease that has devastated citrus crops in Florida.
Citrus greening was first detected in Florida in 2005. Florida has lost approximately $7.8 billion in revenue, 162,200 citrus acres and 7,513 jobs since 2007.
Dean Gabriel, a professor of plant pathology with the UF Institute of Food and Agricultural Sciences and project director, said their work will concentrate on culturing – or growing – the greening bacterium in the laboratory in order to be able to conduct experiments on it. So far, researchers have not been able to do this, creating a major stumbling block to understanding the bacterium and developing treatments.
“People don’t seem to realize the damage this organism has already done to Florida citrus. Orange production dropped more than 50 percent in the last four years, and the pace is quickening,” Gabriel said. “Current treatment testing protocols take years to set up and perform because we can’t culture and rapidly evaluate efficacy. We simply don’t have multiple years left to test new treatments.”
Kathryn Jones, an FSU associate professor of biological science, is also part of the research team.
“I do worry about the general public’s lack of awareness of citrus greening in Florida,” Jones said. “I think there is a general lack of awareness about food production and the challenges related to that. Citrus greening is one of the biggest problems this state is facing right now.”
The disease bacterium first enters a citrus tree via the tiny Asian Citrus Psyllid. When introduced into the plant by leaf feeding, the bacteria then move through the tree via the phloem – the veins of the tree. The disease starves the tree of nutrients, damages its roots and the tree produces fruits that are small, and misshapen, and have reduced quality, making it unsuitable for sale as fresh fruit or, for the most part, juice. Most infected trees eventually become non-productive and the disease has already affected millions of citrus trees in North America.
“There are a number of new approaches to combatting plant diseases, including plant genetic modifications and nanotechnologies,” Gabriel said. “These have been unusually hard to evaluate simply because the causal bacteria cannot be cultured – or grown – in the lab and shipped to other scientists for their research. This team effort is to develop means to rapidly evaluate the efficacy of various approaches against this emerging global threat, not just to citrus but to other crops, including potatoes and tomatoes.”
For her part, Jones will use bacterial viruses to target and manipulate close relatives of that bacterium to hopefully develop a strategy for targeting the bacteria that causes citrus greening.
“I’ve always been interested in plant bacteria interactions,” Jones said. “My focus has always been on genetics and this project builds on past work we’ve done in my lab.”
Other researches involved are: Olufemi Alabi, Texas A&M University; Michael J Davis, University of Florida; Yong-Ping Duan, USDA-Agricultural Research Station, Fort Pierce, Fla., Leonardo De La Fuente, Auburn University; Nabil Killiny-Mansour, University of Florida; Wenbo Ma, University of California, Riverside; Georgios Valadakis, University of California, Riverside; Pamela Roberts, University of Florida; Adriana Castaneda, Instituto Colombiano Agropecuario.