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16.5F: Agrobacterium and Crown Gall Disease - Biology

16.5F: Agrobacterium and Crown Gall Disease - Biology



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Argobacterium causes Crown Gall Disease by transferring a DNA plasmid to the host plant, causing the host to make nutrients for it.

Learning Objectives

  • Summarize the symbiotic relationship between plants and agrobacterium

Key Points

  • Crown Gall Disease is caused by Agrobacterium tumefaciens, a bacteria that infects plants. The bacteria causes tumors on the stem of its host.
  • Agrobacterium tumefaciens manipulates its hosts by transferring a DNA plasmid to the cells of its host. Plasmids are normally used to transfer DNA from bacteria to bacteria.
  • Once in the host cell, the plasmid integrates itself into the host plant cell’s genome and forces the host to produce unique amino acids and other substances which nourish the bacteria. These compounds are unusable by most bacteria, so Argobacteria can out-compete other species.

Key Terms

  • plasmid: A circle of double-stranded DNA that is separate from the chromosomes, which is found in bacteria and protozoa.
  • pilus: A hair-like appendage found on the cell surface of many bacteria.

Crown Gall Disease is caused by a bacteria called Agrobacterium tumefaciens. The disease manifests as a tumor-like growth usually at the junction of the root and shoot. A. tumefaciens can transfer part of its DNA to the host plant, through a plasmid – a bacterial DNA molecule that is independent of a chromosome. The new DNA segment causes the plant to produce unusual amino acids and plant hormones which provide the bacteria with carbon and nitrogen.

Bacteria normally use plasmids for horizontal gene transfer, so they can share genes with related bacteria to help them cope with stressful environments. For example, plasmids can confer on bacteria the ability to fix nitrogen, or to resist antibiotic compounds. Typically bacteria transfer plasmids through conjugation: a donor bacteria creates a tube called a pilus that penetrates the cell wall of the recipient bacteria and the plasmid DNA passes through the tube. The other bacteria either integrates the plasmid into its chromosomes, or it remains free-floating in the cytoplasm. In either case, the recipient bacteria receives new genetic material.

In the case of Crown Gall Disease, A. tumefaciens transfers a plasmid containing T-DNA into the cells of its host plant through conjugation, as it would with another bacteria. However, once inside the plant cell, the DNA integrates semi-randomly into the genome of the plant and changes the behavior of the celll.

The new plasmid genes are expressed by the plant cells, and cause them to secrete enzymes that produce the amino acids octopine or nopaline. It also carries genes for the biosynthesis of the plant hormones, auxin and cytokinins, and for the biosynthesis of opines, providing a carbon and nitrogen source for the bacteria.

These opines can be used by very few other bacteria and give A. tumefaciens a competitive advantage.


Editorial: 𠇊grobacterium biology and its application to transgenic plant production”


  • 1 Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan
  • 2 Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
  • 3 Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan

The extraordinary Agrobacterium research story started from the search for the causative agent of crown gall disease more than 100 years ago. Agrobacterium tumefaciens was first isolated from grapevine galls in 1897 and later isolated from Paris daisy in 1907 (Cavara, 1897a,b Smith and Townsend, 1907). The Agrobacterium infection mechanism involves processing and transfer of a specific DNA fragment (the transferred-DNA, T-DNA) from a bacterial tumor-inducing (Ti) plasmid. Transfer to the plant occurs via a type IV secretion system (T4SS), after which T-DNA is integrated into the plant host genome (Gelvin, 2010 Lacroix and Citovsky, 2013). This interkingdom DNA transfer leads to overproduction of the plant hormones auxin and cytokinin, resulting in tumors. The interkingdom DNA transfer ability of Agrobacterium and the possibility to replace the oncogenes in the T-DNA with genes of interest has made Agrobacterium-mediated transformation the most popular technique to generate transgenic plants.

This Research Topic provides a collection of reviews and original research articles on Agrobacterium genes involved in bacterial physiology/virulence and plant genes involved in transformation and defense against Agrobacterium. A review by Kado (2014) provides a historical overview of how A. tumefaciens was first established as the cause of crown gall disease. In this review, Kado highlights key early plant pathology and milestone molecular biology studies leading to the conclusion that the expression of oncogenes in native T-DNA is the cause of tumor growth in plants. With the solid foundation of these pioneering discoveries, A. tumefaciens evolved from a phytopathogen to a powerful genetic transformation tool for plant biology and biotechnology research.

The first complete genome sequence of an Agrobacterium species (A. tumefaciens C58) was completed in 2001 (Goodner et al., 2001 Wood et al., 2001). The 5.67-megabase genome of this strain carries one circular chromosome, one linear chromosome, and two megaplasmids: the Ti plasmid pTiC58 and a second plasmid, pAtC58. In the review by Platt et al. (2014), the properties, ecology, evolution, and complex interactions of these two A. tumefaciens megaplasmids are discussed. The costs and benefits to A. tumefaciens strains carrying the Ti plasmid and/or the pAtC58 plasmid are discussed and presented from an ecological and evolutionary perspective. Modeling predictions are presented for the relative cost and benefits to A. tumefaciens strains harboring the Ti and/or the pAtC58 plasmids determined by environmental resources. Conjugation and amplification of the Ti plasmid are regulated by the TraI/TraR quorum-sensing (QS) system and conjugal opines. Lang and Faure (2014) review current knowledge of the genetic networks and molecular basis of the A. tumefaciens quorum sensing system. These authors also discuss the biological and ecological impact of the QS system on Ti plasmid conjugation, copy number, and interactions between Agrobacterium and host plants.

During the initial interaction between Agrobacterium and plant cells, bacteria sense various plant-derived signals in the rhizosphere with the help of Ti plasmid-encoded virulence gene (vir gene) and chromosomal virulence gene (chv gene) products. The current knowledge of how A. tumefaciens senses and reacts to different plant-derived signals are summarized in the review article by Subramoni et al. (2014), which also discusses the mechanisms of how the plant hormones auxin, salicylic acid, and ethylene, affect bacterial virulence. Finally, this review discusses the complexity and intricacy of Agrobacterium signaling pathways and the underlying regulatory mechanisms during the initial host cell recognition to maximize subsequent successful infection. In the original research article by Lin et al. (2014), the mechanistic regulation of the membrane sensor VirA protein is further dissected. VirA histidine kinase and the cytoplasmic response regulator VirG protein together play a central role in regulating vir gene expression in response to phenolics. Based on a homology model of the VirA linker region, various mutant and chimeric VirA proteins were generated and examined for their ability to induce VirB promoter activity. The ability of VirA to sense and respond to three separate input signals, phenolics, sugars, and environmental pH, plays a significant role in securing successful infection.

Agrobacterium attachment to plant cells is an important early step in crown gall disease progression. Motile bacteria swim toward host cells and then physically interact with host cells to form aggregates and establish a multicellular bacterial community known as a biofilm. Various genetic and environmental factors that affect Agrobacterium attachment and biofilm formation are reviewed in the article by Heindl et al. (2014). The functions of different types of exopolysaccharides that constitute the biofilm and underlying mechanisms involving how the second messenger cyclic-di-GMP, the ChvG/ChvI system, phosphorus levels, and oxygen tension influence bacterial attachment and virulence are also summarized. In the review article by Matthysse (2014), early studies and current knowledge of the mechanisms of polar and lateral bacterial attachment are summarized. These two mechanisms both contribute to bacterial attachment. When the environmental calcium and phosphate levels and pH values are low, polar attachment predominates. In addition, the phospholipids (PLs), phosphatidylcholine (PC), and phosphate-free lipid ornithine lipids (OLs) contribute to Agrobacterium virulence. In the review by Aktas et al. (2014), the biosynthetic pathways and the physiological roles of these membrane lipids are summarized. The typical eukaryotic membrane lipid PC is not frequently found in bacteria, but it constitutes almost 22% of the Agrobacterium membrane lipid. Interestingly, PCs and OLs may play opposite roles in Agrobacterium virulence. The reduction of tumor formation in a PC-deficient Agrobacterium mutant may result from impaired vir gene expressions controlled by VirA/VirG. The absence of OLs in A. tumefaciens may decrease host defense responses and therefore cause earlier and larger tumor formation.

Plant cells have a variety of receptors that recognize so-called microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs), and subsequently activate plant defense responses, a process known as Pattern-recognition receptor-Triggered Immunity (PTI) (Boller and Felix, 2009 Boller and He, 2009). Agrobacteriium may utilize effectors to hijack plant systems and evade plant defense responses. Pitzschke (2013) reviews strategies used by Agrobacterium to turn plant defense responses to its own advantage. Infected plant cells initiate a mitogen-activated protein kinase signaling cascade that causes VIP1 (Agrobacterium VirE2-interacting protein 1) phosphorylation and translocation into the plant nucleus to induce defense gene expression. On the other hand, Agrobacterium may hijack VIP1 to help T-DNA enter the plant nucleus. Based on the current knowledge of plant defense responses against Agrobacterium infection, Pitzschke (2013) discusses several biotechnological approaches to increase transformation efficiency. In another review by Gohlke and Deeken (2014), early plant responses to Agrobacterium, including various defense responses, hypersensitive responses, and phytohormone level alterations are discussed. The alterations in plant morphology, nutrient translocation, and metabolism caused by crown gall tumor formation are also reviewed. The authors summarize important genomic, epigenomic, transcriptomic, and metabolomic studies that reveal epigenetic changes associated with T-DNA integration and gall development. Subsequently, Hwang et al. (2015) review important pathogenic elicitors, host cell receptor molecules, and their downstream signal transduction pathways in host plants during the PAMP-triggered immune response. They highlight recent discoveries linking plant immunity to endomembrane trafficking and actin dynamic changes. Effects of both the host physiology, including hormone levels, circadian clock, developmental stages, and environmental factors, including light exposure lengths and temperature, on plant defense responses and bacterial virulence are reviewed and discussed.

In nature, evidence of ancient horizontal gene transfers (HGT) from Agrobacterium to plants has been observed in the genera Nicotiana and Linaria. Sequences homologous to mikimopine-type Agrobacterium rhizogenes pRiA4 T-DNA were first discovered in the genome of untransformed tree tobacco, Nicotiana glauca, and named �llular T-DNA” (cT-DNA White et al., 1983). Matveeva and Lutova (2014) review cT-DNA organization, distribution, expression regulation, and a possible correlation with genetic tumor formation in Nicotiana species. They also review recent findings of cT-DNA in the genomes of Linaria species and in other dicotyledonous families. The authors suggest that plants maintaining cT-DNA in their genomes may potentially benefit microorganisms in the rhizosphere by secreting opines in the root zone. They also propose that footprints of ancient pRi T-DNA insertions in the plant genome may provide selective advantage to these plants.

With this Research Topic we provide a platform for scientists to share their understanding of Agrobacterium biology and how Agrobacterium transforms plants. These contributions demonstrate how a highly active research community in plant and microbial sciences can elucidate important pathogenesis questions. Future research on Agrobacteium will continue to advance our understanding of plant-pathogen interactions, and provide new insights useful for plant genetic engineering.


Advanced

Scientific Name
Rhizobium vitis (formerly named Agrobacterium vitis )

Identification
Current season galls

  • First apparent in early summer as swellings on the trunk
  • Soft, convoluted, callus-like tissue, creamy in colour, erupting through the bark layer near injured sites of the vine
  • In young vines, gall formation is often seen just above the graft union
  • By late summer the galls darken and become corky in texture with a rough surface and persist for several years
  • Dead galls may flake off the vine
  • Young galls may often form at the periphery of old galls
  • Typically seen from the soil line to the first wire
  • Stressed infected vines are often killed during low temperature episodes in the winter

Often Confused With:
Excessive callusing of nursery stock under wet conditions: callus does not become corky and flake off.

Biology
The crown gall bacterium survives within galls and systematically infested vines. The bacterium remains inside the vine, without causing symptoms, until there is an injury to the trunk and only then invades the outer part of the trunk where it causes rapid cell multiplication and distortion of tissue producing galls. The crown gall bacterium may also survive in vineyard soils in vine debris. It is believed that the majority of infections are a result of symptomless contaminated planting material. Generally, the incidence of crown gall is correlated with cold susceptibility less cold tolerant varieties having a higher incidence of crown gall infection.

Period of Activity
Early summer, particularly after winter injury has occurred in cold sensitive varieties.

Scouting Notes
Galls are mostly found on the lower trunk, from the soil line to the first wire however, aerial galls may develop more than one metre up the trellis. Monitor these areas of the trunk for galls starting in early summer. Severely diseased vines usually exhibit significant reductions in yield and vigour, predisposing them to winter kill.

Threshold
There is no threshold. Trunks with crown gall symptoms will weaken and die. However, other symptomless trunks on the same vine, while infested, may continue to produce crop for many years. If the gall is at the graft union and no suckers develop, the vine will die.

Management Notes
Management practices that reduce injury are important in managing this disease, since the expression of crown gall is closely correlated with the occurence of injury.

Before Planting

Losses of grape plants due to crown gall may be minimized with some considerations before vineyard site selection or planting.

  • Select sites with good soil and air drainage, avoid frost-prone areas
  • Select rootstocks that are resistant to crown gall such as Courderc 3309, 101-14 Mgt, and Riparia Gloire,
  • Select hardy, cold tolerant varieties where possible
  • Do not replant old vineyard areas where crown gall was present less than 2 years after grapevines have been removed. Crown gall bacteria can survive in the remnants of the old grape plants until the debris decomposes. When removing diseased vines, remove as much of the root system as possible.
  • Purchase vines from a reputable source. Latently-infested nursery stock is the major source of crown gall disease in vineyards.
  • Hot water treatment of vines is effective in reducing crown gall infection levels in planting materials.

After Planting

There is little that can be done to control this disease once it is established in the vineyard other than to avoid injury to vines (winter, mechanical and human) that will activate the disease.


Advanced

Scientific Name
Agrobacterium tumefaciens

Identification

  • Galls on roots, crowns, and occasionally trunks and scaffolds
  • Galls are spherical, lumpy and rough, varying from 1 to more than 10 cm in diameter
  • Galls are initially soft and smooth but turn dark, hard, rough, woody and cracked as they enlarge and age
  • Galls generally occur only on one side of the root
  • Young trees can be girdled and killed fairly quickly by crown galls Galls are usually not serious on older trees unless they are invaded by wood decay fungi

Often Confused With
Root knot nematode galling on roots - swelling occurs across the entire diameter of the root rather than just on one side

Biology
The pathogen affects a wide range of broadleaf, woody plants, including stone fruits. Bacteria are released into the soil when galls are wet or when older gall tissue disintegrates. The bacterium can survive in the soil for at least 1 year in the absence of host tissue. Established trees are infected only through wounds, such as those caused by growth cracks, pruning, damage from cultivation equipment or freezing injury. Seedlings can be infected during germination if planted into infested soil. The galls interfere with the normal flow of water and nutrients. Young trees may be killed while older trees suffer reduced growth and vigour.

Bacteria enter the roots and crown through wounds produced in caring for, and handling the nursery stock. They may also enter through wounds made by root feeding insects. Following infection, crown gall bacteria invade the host tissue, multiplying between host cells. A portion of the bacteria’s genetic material becomes incorporated into that of the host cells, causing them to proliferate and produce unusual amino acids that serve as a food source for the bacteria. The proliferation of these cells results in gall formation.

Symptoms may develop in a few weeks at moderate temperatures or the bacterium may remain latent for 2-5 years before symptoms are produced. If crown gall occurs in the nursery, symptoms are usually well developed on finished trees at the time of digging.

In addition to primary galls, secondary galls sometimes develop at some distance from the initial infection. These galls may develop on unwounded tissue and the bacteria cannot be found associated with them.

Period of Activity
Symptoms may develop in a few weeks at moderate temperatures or the bacterium may remain latent for 2-5 years before symptoms are produced.

Thresholds
There is no tolerance for crown gall infested nursery trees.

Scouting Notes
Inspect nursery trees for signs of galls before planting. Monitor young trees for signs of collapse and older trees for loss of vigour. Check roots, crowns , trunks and scaffolds for crown gall.

Management Notes
Plant in well-drained fields and rotate contaminated field sites with non-host plants such as grasses or grains.

In the nursery, a sterile planting medium should be used.

Use only crown gall-free nursery stock from a reputable nursery. Carefully inspect nursery stock before planting and return the entire lot if symptomatic trees are found. Plant seedlings with little or no heeling in.

Handle young trees to avoid injury as much as possible, both at planting and during the life of the tree in the orchard.

Remove trees found with large galls surrounding the crowns when the trees become unproductive.

When replanting a previously affected site, remove as many of the old tree roots as possible, grow a grass rotation crop to help degrade leftover host material and reduce pathogen levels, and offset the new trees from the previous tree spacing to minimize contact of healthy new roots with any infested roots that may remain.


16.5F: Agrobacterium and Crown Gall Disease - Biology

Agrobacterium tumefaciens
By Alyssa Collins
A Class Project for
PP728 Soilborne Plant Pathogens
North Carolina State University
Department of Plant Pathology

Agrobacterium tumefaciens, the cause of the economically important disease, crown gall, has also been studied for years because of its remarkable biology. The mechanism this bacterium uses to parasitize plant tissue involves the integration of some of its own DNA into the host genome resulting in unsightly tumors and changes in plant metabolism. A. tumefaciens prompted the first successful development of a biological control agent and is now used as a tool for engineering desired genes into plants.

Host Range and Distribution

Agrobacterium tumefaciens is cosmopolitan in distribution, affecting dicotyledonous plants in more than 60 different plant families. Crown gall can be found most often on stone fruit and pome trees as well as brambles and several species of ornamental plants.

Agrobacterium tumefaciens is a member of the family Rhizobiaceae. These bacteria are Gram-negative and grow aerobically, without forming endospores. The cells are rod-shaped and motile, having one to six peritrichous flagella. Cells are 0.6-1.0 m m by 1.5- 3.0 m m and may exist singly or in pairs. In culture on carbohydrate-containing media, cells produce large amounts of extracellular polysaccharides, giving colonies a voluminous, slimy appearance.

Recently, a reclassification of the species of Agrobacterium has been undertaken by use of ribosomal RNA sequencing as a taxonomic tool. The resulting nomenclature places the former species, A. tumefacians biovar 1, A. radiobacter biovar 1, and A. rhizogenes biovar 1, within the new taxon: Agrobacterium tumefaciens.
Isolation
A. tumefaciens can be effectively isolated for identification from gall tissue, soil or water. Optimal gall tissue for isolation is white or cream-colored from a young, actively growing gall. The gall should be washed or surface sterilized using 20% household bleach, and rinsed several times in sterile water. Cut a few samples from different parts of the white tissue of the gall, and further divide samples into small pieces. Place these pieces into a culture tube containing sterile distilled water or buffer, vortex and allow to stand for at least 30 minutes. Using an inoculating loop, streak this suspension on Medium 1A (Schaad et al., 2001), and incubate at 25-27 ° C. Different strains will grow at different rates. One may also use this selective medium to detect A. tumefaciens in soil dilutions or irrigation water.

It should be noted, however, that the presence of A. tumefaciens cells in a sample does not necessarily dictate the existence of the crown gall-inciting strain in the sample. Only cells containing a specific plasmid (the Tiplasmid) can cause disease. A. tumefaciens strains lacking the plasmid live as rhizosphere-inhabiting bacteria without causing disease.

Symptoms

Crown gall manifests itself initially as small swellings on the root or stem near the soil line, and occasionally on aerial portions of the plant. Young tumors, which often resemble the callus tissue that results from wounding, are soft, somewhat spherical and white to cream colored. As tumors become older, their shape becomes quite irregular, and they turn brown or black. Tumors may be connected to the host surface by only a narrow bit of tissue, or may appear as a swelling of the stem, not distinctly separate. The tissue can be spongy and crumbling throughout the gall or can be woody and knot-like. Several tumors may occur on the same plant and may rot from the surface of the plant completely or partially, possibly developing repeatedly in the same area season after season. Additional symptoms include stunting, chlorotic leaves, and plants may be more susceptible to adverse environmental conditions and secondary infection.

Pathogenic strains of A. tumefaciens may live saprophytically in soil for up to two years. When a nearby host plant is wounded near the soil line by insect feeding, transplant injury or any other means the bacterium chemotactically moves into the wound site and between host cells. These bacteria then stimulate the surrounding host cells to rapidly and irregularly divide. The bacterium accomplishes this by inserting a piece of its own DNA into the host cells' chromosomes, causing overproduction of cytokinins and auxins which are plant growth regulators, and opines which serve as nutrients for the pathogen. The resulting tissue is undifferentiated with a white or cream color, and cells may have one or more nuclei. This tissue continues to enlarge and a tumor is formed on the root or stem of the plant, depending on original wound site. The bacteria occupy the intercellular spaces around the periphery of the gall and are not found in the center of the enlarging tumor. The tumor is not protected by an epidermis, leaving the tissue susceptible to secondary pathogens, insects and saprophytes. Degradation of the tumor by secondary invaders causes brown or black discoloration and releases A. tumefaciens cells back into the soil to be carried away by with soil or water, or remain in the soil until the next growing season. In perennial plants, part of the infected tissue may remain alive and inhabited by A. tumefaciens, which, even if the tumor has sloughed off, can persist to cause a new tumor the following season in the same place.

Introduction of pathogenic A. tumefaciens strains can be avoided by thorough inspection of nursery stock for crown gall symptoms. Susceptible varieties should not be planted in soils known to be infested with the pathogen. These soils should be planted in a monocotyledonous crop like corn or wheat for several years. Nursery stock should be certified crown gall-free and should be budded rather than grafted. If the threat of crown gall exists, all practices that wound tissue should be avoided and chewing insects should be controlled.

Preventative treatment of seeds or transplants with the non-pathogenic biocontrol organism Agrobacterium radiobacter is a relatively inexpensive and effective means of managing the development of crown gall in commercial operations. Application of this antagonist by soaking seeds or dipping transplants can prevent infection by most strains of A. tumefaciens due to the production of the antibiotic agrocin 84 by strain K84 of A. radiobacter. Some curative properties are exhibited by a commercially available mixture of 2,4-xylenol and metacresol in an oil-water emulsion when painted directly on established tumors. But this is rarely used due to labor and time constraints.

Agrios, G.N. 1988. Plant Pathology, 3 rd Ed. Academic Press Inc., London. pp. 558-565.

Horst, R.K. 1983. Compendium of Rose Diseases. APS Press, St. Paul, MN. pp 23-25.

Schaad, N.W., J.B. Jones & W. Chun. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 3 rd Ed. APS Press, St. Paul, MN. pp. 17-35.

Links to other sites with information about Agrobacterium tumefaciens


Crown Gall Disease of Nursery Crops

Note all the galls along the stem to the right. Many have started at pruning wounds.

L.W. Moore (deceased), Bacteriologist and Plant Pathologist, OSU

Updated by M. L. Putnam, Diagnostician and Plant Pathologist, OSU

Crown gall continues to be a major problem for the nursery industry, both in woody and herbaceous plants. The pathogen traditionally known to cause crown gall in the most plants is Agrobacterium tumefaciens ( Rhizobium radiobacter ). The pathogen name has been under dispute for decades, and A. tumefaciens is known to be a species complex, consisting of at least 11 different genomospecies. Here we will refer to the bacteria that cause crown gall as tumorigenic agrobacteria. Other species of Agrobacterium can also cause galls: A. rubi is much less common named for the host in which it was first found ( Rubus spp.), it has since been found in galls on rose, and will likely be found in other plants with time. Agrobacterium vitis (= Allorhizobium vitis ) causes galls on grapevines. A. larrymoorei causes galling of Ficus benjamina , and has recently been found in rose galls. A novel gall-forming species, also isolated from rose, was recently described and named A. rosae . It is likely that additional species will be named in coming years, as bacteria associated with galls are examined more closely using modern molecular techniques. All of these species have a similar biology. This discussion covers the biology, host range, symptoms, and management of the disease.

Crown gall is a tumor-forming disease of plants caused by tumorigenic agrobacteria, many of which are thought to be present in most agricultural soils. The pathogens, in soil or on infested plants, are disseminated by splashing rain, irrigation water, heeling-in galled plants with healthy plants, farm machinery, pruning tools, wind, and plant parts used for propagation. Wounds are required for the pathogen to infect a plant. Wounds are made by pruning and cultivation, emergence of lateral roots, frost injury, and insect and nematode feeding. The pathogen colonizes the wound, attaches firmly to injured plant cells, and transfers part of its DNA into the DNA of the plant. Galls appear in a matter of weeks at temperatures above 70°F on herbaceous plants woody plants such as roses may not show galls until months or years after exposure. Latent infections typically develop into galls in a later growing season. Pathogenic bacteria can be shed from the gall into the surrounding soil or water where they colonize or infect new plant tissues.

Although commonly reported to have a host range of hundreds, this information is based on artificial inoculations often of just a single isolate. On a practical basis, far fewer plants are naturally susceptible. (Examples of host plants infected by Agrobacterium are listed in Table 3.) However, the root systems of non-host plants such as weeds, grasses, and cereals can harbor the pathogen and serve as a reservoir of inoculum in natural settings.

The disease is called crown gall, but galling may be found at the base of cuttings, on roots, crowns, or on stems, canes, vines, or leaves. Leaf galls are usually found on herbaceous plants that have a systemic infection. (Herbaceous ornamental plants susceptible to crown gall are shown in Table 1.) Galls often occur at pruning wounds. Galls are usually rounded and may be smooth or textured like a cauliflower head. On woody perennial plants, galls become more woody and fissured with age, sometimes reaching a diameter of 6 inches, and girdling the stem. Galls on grapevines, blueberry, and bramble fruit are usually elongate, erumpent ridges of tissue bursting through the outer stem tissues.

Woody plants infected the first year they are planted out are more severely damaged. (Woody plants susceptible to crown gall are shown in Table 2.) Severely galled young plants are weakened, stunted, and unproductive and occasionally die due to an inferior root system. Literature reports of crown gall damage are contradictory they range from benign to debilitating to deadly.

Symptoms become evident 2 to 4 weeks after infection if temperatures are at or above 68°F, usually coinciding with warmer soil temperatures in May or June. Initially, the galls look like callus outgrowths but then increase rapidly in size and number. Symptom development slows greatly below 58°F and stops below 50°F. Infection is inhibited above 92°F to 95°F. Latent infections are symptomless and usually occur when soils are cool. Gall symptoms typically develop at the infected wound the following season on rare occasions galls don’t appear until the third growing season.

Some problems can look like crown gall but are not pathogenic. Aerial burr knot on apple tree trunks and branches is a cushion-like assemblage of adventitious roots its cause is thought to be genetic rather than an infectious agent.

Small galls require careful diagnosis because they may be confused with excessive wound callus. Detection using molecular methods specific to plasmid gene regions involved with virulence, or isolation of bacteria later identified as pathogenic is necessary to confirm a crown gall diagnosis. Nonpathogenic Agrobacterium cells are often prevalent in these same tissues and can reach high populations. That makes diagnosis difficult, especially in galls on apple, blueberry, and grapevines where non-pathogens can constitute over 99% of the Agrobacterium population.

Disease Management – Woody Nursery Stock

Pathogen-free plants grown in uninfested soil will not develop crown gall. This emphasizes the importance of planting clean propagating material in clean soil. Good sanitation and cultural practices are important deterrents to crown gall. Discard all nursery stock showing symptoms to avoid contaminating healthy plants and storage facilities. At harvest, leave noticeably galled plants in the field for later pickup and destruction. If possible, choose a rootstock that is less susceptible, avoid planting sites heavily infested by root-attacking insects and nematodes, disinfect pruning equipment between trees, and adopt management practices that minimize wounding. Avoid planting into heavy, wet soil. Don’t plant trees deeper than they grew in the nursery. If possible, incubate dormant seedling roots at 73°F to 76°F for 10 to 14 days to heal wounds and reduce susceptibility to tumorigenic agrobacteria before planting them in wet soil. Use irrigation water from wells, if possible. Avoid planting where galled plants grew in the last 4 to 5 years choose fields that were planted recently to vegetables or grain. In summary, think prevention —avoid exposing plants to tumorigenic agrobacteria at any stage of plant production.

Crown gall is generally much more prevalent in heavy soils or in soil where water stands for a day or so. In New York, crown gall incidence was highest on a heavy clay knoll (15 ft elevation) from which water drained toward flat, loamy portions of the field. In Oregon, gall incidence on an Old Home x Farmingdale pear rootstock selection was severe (495 of 500 trees infected) in a heavy, wet soil, but in the same field only 1 of 500 trees was galled outside the wet area.

Cropping history can influence crown gall incidence. Budded apple trees became badly galled in fields where a previous nursery crop such as grape, peach, raspberry, and rose had been heavily infected. This situation isn’t repeated at every site, but we still recommend avoiding fields with a recent history of crown gall.

Reports of resistance in plants normally susceptible to crown gall are limited and depend on the strains of bacteria present in a given location. There are no reliable lists of cultivars with resistance that hold up in all geographic locations. It is better to select plants that are not susceptible in the first place if crown gall is a chronic problem in a particular field.

Using A. radiobacter K84, a biological control agent, has been very effective against crown gall on a number of hosts, but exceptions exist. Strain K84 produces a toxin against some tumorigenic strains of agrobacteria. This biological control is solely preventive, not curative application timing is critical to properly protect plant wounds caused at harvest or by pruning. Htay and Kerr recommend seed and root treatment with K84 for best results. Not all strains of tumorigenic agrobacteria are sensitive to K84. For example, most agrobacteria isolated from grape tumors are A. vitis , which are insensitive to K84. If K84 has been used properly and galling persists, its use should be discontinued since it is likely the bacteria present are not sensitive to the product.

An improved, genetically engineered strain of K84 called K1026 is available. Its use is preferable, since the K1026 bacteria are not capable of transferring to other bacteria the genes that produce the toxin.

Biological control is compatible with a few pesticides such as metalaxy (Ridomil), thiram and thiophanate-methyl (Topsin) but not with captan, etridiazole alone (Truban), etridiazole plus thiophanate-methyl (Banrot or Zyban), mancozeb, PCNB or streptomycin. It is also not compatible with chlorinated water.

No registered chemicals that effectively control crown gall are currently available in the United States. In general, chemical preplant dips or soil drenches have been ineffective.

Fumigation to rid soil of Agrobacterium generally has been ineffective, and in some cases, growers reported more disease after fumigation.

Heat therapy has been tried in cherry and plum seedlings, and in dormant grape cuttings. Although these measures can reduce the incidence of disease, there will still be a small percentage of plants that remain infected. Time and temperatures needed for effective heat therapy has not been determined for many plants, and injury to the plant material can occur when temperatures are too high. Although promising, heat therapy is not commonly used due to these difficulties.

In solarization, a thin plastic film is stretched over moist soil to capture energy from the sun and heat the soil to temperatures that kill pathogenic microbes. Populations of tumorigenic agrobacteria could not be detected in a solarized sandy loam soil, but solarization did not work in the heavier silty-loam. Mazzard cherry seedlings planted later in solarized and in nonsolarized control plots developed crown gall only in the nonsolarized plots.

Following is a summary of the best practices for managing crown gall. They include experimental results and grower observations. Understandably, physical and economic constraints occasionally may impede applying all these practices. But for best results, follow or adapt the procedures as closely as possible to fit your management plan.

Best Practices for Managing Crown Gall

  • Discard diseased plants as soon as noticed to avoid cross-contaminating other plants, equipment, or storage facilities.
  • Don’t heel-in galled plants with healthy plants.
  • Use good sanitation in handling planting stock.
  • Minimize wounding disinfect pruning tools between plants.
  • Plant only disease-free stock.
  • Plant in clean soil.
  • Avoid fields with a recent history of high crown gall infestation.
  • Avoid fields with heavy infestations of root-attacking insects and nematodes.
  • Select well-drained soils tile heavy soils.
  • Field-fallowing is helpful but may be impractical west of the Cascade Range.
  • Rotate susceptible crops with small grains.
  • Plant when soil is below 50°F.
  • Solarize lighter soils.
  • Avoid mechanical injury from tillage, hoeing.
  • Irrigate with deep-well water or sanitized pond water.
  • Keep grafts and buds above soil line.
  • Avoid high nitrogen and irrigation late in the growing season.

The following are specific procedures for commonly grown plants that can be used in addition to the above general procedures.

Stone Fruit, Nut Crops, Roses: Dip or spray with the biocontrol agent K84 or K1026. Apply to seed, bare roots, and aboveground grafts.

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Cazelles, O., and Epard, S. et al. 1991. The effect of disinfection with oxyquinoline sulfate of the Berl. x Rip. 5C rootstock on the expression of crown gall in grape propagation. Revue Suisse de Viticulture, d’Arboriculture et d’Horticulture 23:285-288.

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Background

Crown gall disease was identified long ago as a bacterial plant disease [1], and its pathogenic bacterium is Agrobacterium tumefaciens, which mainly infects dicots. This disease often results in severe economic losses to the production of cherry and other fruit trees [2,3,4]. Crown gall disease starts with the attachment of A. tumefaciens to plant cell. And then the transfer DNA, a portion of the Ti plasmid, will be integrated into the plant genome. Finally, the symptomatic tumors form and grow [5].

Crown gall disease affects many fruit trees and causes extensive economic losses in nurseries. In a previous study, 11 tree species were surveyed. The highest disease incidence was found in peach (Prunus persica [L.] Batsch), almond (P. dulcis D Webb), cherry (P. avium L.), apple (Malus sylvestris Mill) and olive (Olea europaea L.) [6]. It was also found the rootstock of peach, cherry, apple and pear (Pyrus communis L.) trees was a influence factor contributing to the significant differences in the frequency of galled plants.

Plants are often exposed to many various bacterial, viral, and fungal pathogens but have evolved potent defense systems to protect themselves [7]. In defense responses of plants, the identification of microbial pathogens plays a key role, as it “turns on” the signal transduction pathway which activates the expression of numerous pathogen-responsive genes [8, 9]. These disease resistance genes are crucial for identifying the effector proteins during the process of pathogen infection [7].

Many biotechnological strategies have been developed and applied in the attempt to control crown gall disease. In transformation experiments, the truncated genes involved in T-DNA transfer have been used to induce plant resistance to crown gall disease [10, 11], and inactivating the oncogenes could prevent tumor formation [12]. Therefore, to obtain plants that are resistant to crown gall disease, much research has been devoted to producing sense and antisense strands of the oncogene sequence by placing these sequences between opposing strong constitutive promoters [13], or to silencing the involved bacterial oncogenes by using premature stop codons [14]. The study of Niemeyer et al. (2014) demonstrated a successful reprogramming of the viral N gene response against crown gall disease [9]. In recent years, Rosalia Deeken’s group has been working on the molecular mechanism between crown gall disease and A. tumefaciens in Arabidopsis thaliana [8, 15,16,17,18]. Pathogen infection always induces response of plant hormones. Lee et al. (2009) explored the physiological changes and adaptations on the aspect of SA, JA, ethylene (ET), and auxin (indole-3-acetic acid, IAA) with changes in the Arabidopsis thaliana transcriptome during tumor development [5].

At present, planting resistant cultivars and developing biological antagonists both are effective measures to control crown gall disease in orchards [3]. The existing biological antagonists are mainly used for prevention but they act poorly as a treatment. So the crown gall-resistant cultivars in agriculture were in need [19]. Previous studies have reported crown gall-resistant cultivars for apple, peach, plum, grapevine, aspen, and roses [20,21,22,23,24,25,26,27]. Crown gall resistance has been assessed in accessions of 20 Prunus species [21]. And it was found that when the strains K12 and C58 of A. tumefaciens were used to infect the main stems or lateral branches of seedlings, the incidence of resistance was up to 30% in some accessions of P. mahaleb. The cherry breeding resource plant P. mahaleb is a cosmopolitan cherry rootstock. In northwest China, it has become one of the main sweet cherry rootstocks because of its excellent biological traits, such as strong resistance to crown gall disease, dwarfing ability and salinity among other desirable traits [28]. By systematic classification of cherry species, P. mahaleb belongs to the III. Cerasus subgenus, Section 5 Mahaleb Focke [29]. It is a deciduous tree or large shrub, growing to 2–10 m (rarely up to 12 m) tall with a trunk up to 40 cm diameter. In most cherry growing countries, mahaleb cherry is used to be rootstock of sweet and sour cherries [28]. This rootstock showed strong resistance to crown gall disease in cherry production, but little is known about its mechanism of crown gall resistance. Furthermore, the actual genes (without modification) underpinning resistance to crown gall have not yet been reported.

In this study, we focused on cherry rootstock ‘CDR-1’ (P. mahaleb), the natural hybrid cultivar of P. mahaleb. The objective of our study was to investigate the resistance mechanism of ‘CDR-1’ to crown gall disease. Here, we carried out morphological observations, physiological and biochemical analyses, gene expression analysis and transcriptomic analysis in ‘CDR-1’, and conducted transient expression and transgenic verification in tobacco. Our results provide evidence that the crown gall resistance of ‘CDR-1’ is likely related to the lignin biosynthetic pathway.


Experimental Procedures

Plant materials

Arabidopsis thaliana ecotype Col-0 was used for the seedling transformation assay, transcriptome assay and Agrobacterium inoculation assay. GS mutants in the Col-0 background, including myb28/myb29 (SALK_136312 x GABI_868E02), cyp81F2-1 (SALK_073776), cyp81F2-2 (SALK_123882), myb51-1 (SM_3_16332), myb51-2 (SALK_059765), cyp79B2/cyp79B3 (Zhao et al., 2002 ), pen2-1 (Lipka et al., 2005 ) and pen2-2 (GABI-KAT 134C04), the camalexin mutants, including cyp71A12 (GABI-KAT 127-H03), cyp71A13-1 (SALK_105136), cyp71A13-3 (SALK_128994) and pad3-1 (CS3805), and the cyp79b2/B3/myb28/29 quadruple (qko) mutant, completely free of GSs and camalexin, were used in the transient seedling transformation assay as described.

Agrobacterium transformation of Arabidopsis seedlings and GUS assays

The virulent A. tumefaciens wild-type strain C58 was used for the infection of Col-0 seedlings. Seeds were germinated in 2 mL of half-strength Murashige and Skoog (MS) (Basal Salt Mixture, PhytoTechnology Laboratories, Kansas City, Kansas, USA) liquid medium [half-strength MS salt supplemented with 0.5% sucrose (w/v), pH 5.7] in each well of a six-well plate. Germination and growth took place in a growth room at 22 °C under a 16-h/8-h light–dark cycle (100 µmol/m 2 /s). Virulence of A. tumefaciens was pre-induced by 200 µ m acetosyringone in AB-MES (AB Minimal Medium plus MES salt, pH 5.5) (Wu et al., 2014 ) at 25 °C for 16 h prior to infection. The Arabidopsis seedlings were infected with pre-induced A. tumefaciens C58 cells at an optical density at 600 nm (OD600) = 0.02 in half-strength MS medium. If the removal of agrobacterial cells was necessary, co-cultivation medium was removed after the chosen infection time and replaced with 2 mL of freshly prepared half-strength MS medium containing 100 µ m timentin, and incubated for recovery before analysis.

For the monitoring of the transient transformation efficiency, the T-DNA vector pBISN1 carrying the gusA-intron genes (Narasimhulu et al., 1996 ) was transformed into A. tumefaciens strain C58 for infection of Arabidopsis seedlings. GUS staining and activity assays were carried out as described at the chosen infection time (Salinas and Sánchez-Serrano, 2006 Wu et al., 2014 ). In brief, seedlings were stained by incubation in GUS staining solution containing 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc), and incubated at 37 °C in the dark overnight, followed by destaining in 90% ethanol (EtOH). For the GUS activity assay, liquid nitrogen-frozen seedlings from each well were ground into a fine powder to extract total protein. The GUS activity in 20 µg of protein per 200-μL reaction was quantified with the fluorescence substrate 4-methylumbelliferyl-β- d -glucuronide (MUG). The fluorescence intensity (excitation, 365 nm emission, 455 nm filter at 430 nm) was measured using a Microplate Reader (BioTek, Taipei, Taiwan) at 37 °C for 1 h. GUS activity was normalized to the protein amount and 4-methylumbelliferone standard curve. For statistical analysis, one-way analysis of variance (ANOVA) with Dunnett's test was performed. To determine the effects of GS-derived metabolites and camalexin on GUS enzyme activity in vitro, the selected compounds and DMSO control were each incubated with 5 ng of recombinant GUS protein (Sigma-Aldrich, St. Louis, MO, USA) in GUS extraction buffer containing 1 m m MUG. The reaction mixture was measured for GUS activity at 37 °C for 1 h.

Transcriptome analysis

For gene expression profiling of Agrobacterium-infected seedlings, the shoots and roots of Col-0 seedlings (infected or mock control) were separated by cutting with a micro-scissor and immediately frozen in liquid nitrogen. Total RNA was extracted according to the phenol (pH 4.5)/chloroform protocol, followed by gene expression analysis with Affymetrix ATH1 chips (Affymetrix, Santa Clara, CA, USA). The chips of three biological repeats were normalized by the MAS5.0 algorithm using GeneSpring software (Agilent Technologies, Santa Clara, CA, USA), and the genes with an intensity higher than the background value (value > 75), which passed the asymptotic unpaired t-test with Benjamini–Hochberg test correction (FDR, P < 0.05), were selected for further analysis. The fold changes were determined from the signals of infected plant tissues versus mock infection controls under the same conditions, and two-fold changes were used as cut-off to determine Agrobacterium-responsive genes. GOBU software (Lin et al., 2006 ) was used to analyse GO. The significant GO items were calculated with elim Fisher's exact test (P < 0.01) based on gene counts (Alexa et al., 2006 ).

Crown gall growth assay

For the crown gall growth assay, the A. thaliana wild-type Col-0 and mutants were cultivated in growth cabinets (Percival, CLF, Wertingen, Germany) under short-day conditions at 22 °C (8 h of 80–100 µmol/m 2 /s light Osram 400 W, Power Star HQI-E 400W/DV, 380–780 nm) (Wuerzburg, Germany) and 16 °C during the dark period (16 h) with a relative humidity of 50%–60%. Tumour development was induced by streaking virulent A. tumefaciens strain C58 into a wound of 1.5 cm in length, scratched into the base of young 5-cm-long inflorescence stalks. Tumour tissue was harvested 28 days after infection using a scalpel and a binocular. Wounded, but uninfected, tumour-free inflorescence stalk sections of the same age served as reference tissues.

GS and camalexin analysis in Arabidopsis seedlings

Extraction and analysis of seedling GSs and camalexin were performed and modified as described previously (Glauser et al., 2012 Zandalinas et al., 2012 ). In total, 100 mg FW of Arabidopsis seedlings were homogenized and dissolved in 1 mL of 70% high-performance liquid chromatography (HPLC)-grade methanol containing 12.5 ng/μL sinalbin (4-hydroxybenzyl GS) as an internal standard. The supernatants obtained were heated at 80 °C for 20 min and subjected to a UPLC-Synapt G1 high-definition mass spectrometry (HDMS) system (Waters, Taipei, Taiwan). GSs were separated on an Acquity CSH C18 column (length, 100 mm 2.1 mm i.d. 1.7 μm Waters) at a flow rate of 400 μL/min. The GSs were eluted by solvent A (2% acetonitrile and 0.05% formic acid) and solvent B (100% acetonitrile and 0.05% formic acid) for 8 min in 1%–45% solvent B and 1 min in 45%–100% solvent B. The fractions were injected for MS analysis, and negative ion data were recorded in MS1 mode. The peak area was calculated by MassLynx software (Waters), and then normalized to nanomoles for GSs or micrograms for camalexin per gram FW. The GSs were quantified with the given references, including I3M for iGSs, 4MTB for methylthioalkyl GSs and 4MSOB for methylsulfinylalkyl GSs, purchased from AppliChem (Darmstadt, Germany). Camalexin was quantified with pure camalexin (Sigma-Aldrich, St. Louis, MO, USA).

GS and camalexin analysis in Arabidopsis inflorescence stalks

For GS analysis of infected Arabidopsis inflorescence stalks, 100 mg (FW) were lyophilized, thoroughly homogenized and extracted three times with 1 mL of 80% (v/v) methanol. For the first extraction step, benzyl GS (Phytoplan, Heidelberg, Germany) was added to each sample as internal standard. GSs were desulfonated as described previously (Agerbirk et al., 2001 ), and separated on a Grom-Sil 80 ODS 7 pH column (length, 60 mm 4 mm i.d. 4 μm Alltech) (Wuerzburg, Germany) by HPLC (Agilent 1200, Waldbronn, Germany flow rate, 0.25 mL/min). The desulfo GSs were eluted as follows: 0.3 min in 0%–5% solvent A (water), 7 min with 1.2 min hold in 5%–95% solvent B (methanol) and 3.5 min in 95%–5% solvent B. Desulfo-GSs were determined via UV diode array detection (229 nm), identified and quantified using particular response factors (aGSs, 1 iGSs, 0.26) (Gonzáles-Megías and Müller, 2010 ).

Camalexin was extracted from lyophilized tissue (50 mg FW) by the addition of 400 μL of 85% methanol. The samples were thoroughly homogenized with a metal ball in a Mixer Mill 301 (Retsch, Haan, Germany) for 1.5 min at a frequency of 30 Hz. The extract was incubated at 42 °C for 60 min with addition of 0.3 μg/μL camalexin as an external standard. For the identification and quantification of camalexin, HPLC was applied as described by Mikkelsen et al. ( 2009 ).

GS derived metabolites and camalexin treatment for transient transformation assays and Agrobacterium cell counts

The selected aGS-ITCs (LKT Laboratories, St Paul, MN, USA) and camalexin were dissolved in DMSO, and I3M was dissolved in methanol. These compounds were added to the seedling co-cultivation medium for Agrobacterium infection and GUS assays, as described above.

For the measurement of the viable Agrobacterium cell number, the bacterial cells (C58 strain carrying pBISNI) in co-cultivation medium and associated with seedlings at 1 and 3 dpi were collected. Six seedlings per well were washed by 2 mL of double-distilled H2O to remove unbound bacteria and ground by a mortar in 1 mL of 0.9% NaCl solution. The bacterial cells in medium or associated with seedlings were 10× serially diluted and then plated on 523 medium (Kado and Heskett, 1970 ) containing kanamycin, and incubated at 25 ºC for 2 days to obtain colony-forming units (CFUs). The seedling-associated Agrobacterium cell number was further normalized to the plant fresh weight.

Myrosinase activity

Myrosinase activity was determined from 50–200 mg of frozen plant material, which was purified from internal substrate. Activity was measured by the photometric quantification of the released glucose from standardized amounts of externally added substrate according to the protocol developed by Travers-Martin et al. ( 2008 ).

Callus induction assay

Callus induction assay was performed and modified as described previously (Hwang and Gelvin, 2004 ). Col-0 and the tested mutants were grown on half-strength MS agar plates for 3 weeks, and the roots were cut into ∼1-cm segments. About 60 root explants were transferred to agar plates containing callus induction medium (CIM), further incubated for 4 weeks, followed by counting of the number of developing calli and calculation of the rate of callus induction.


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