Functional and Applied Genomics of Fruit Crops Research Project

Background - Fruit trees, to be productive, require favorable environments, both biotic and abiotic.  Among abiotic stresses, acute freeze damage can eliminate entire orchards, and drought stress is a continual and serious threat to US agriculture that causes billion-dollar losses.  Even trees that survive freezing temperatures or drought have reduced vigor, longevity, and productivity. As an example, it was estimated that in New York State alone, 25,000 apple trees were lost to winter damage in 2003-2004 at a valued cost of 2.5 million dollars.  Annual losses in the U.S. for all crops are estimated to be almost one billion dollars. Similarly, between 1978 and 1995, average crop losses due to drought in the US exceeded $1.2 billion each year.  Since 1995, the US has experienced 3 major droughts with losses estimated at over 1 billion dollars for each occurrence.  Recent drought monitoring results suggest that the US West Coast drought will continue to affect crops in the northwestern states.  Among diseases, fire blight is easily transmitted, difficult to control, and can be catastrophic.  A single fire blight epidemic that occurred in southwest Michigan in 2000 was estimated to have caused the death of 350,000 to 450,000 trees and the removal of 1,500 to 2,300 acres of apple orchards resulting in an economic loss of $42 million. Streptomycin is the primary control agent, but resistant strains of the pathogen, Erwinia amylovora, are appearing in some areas. 

Genomic analysis of herbaceous annual model plants, especially Arabidopsis, has to some degree elucidated the molecular basis of processes to overcome these problems.  Genomic knowledge, including gene identification and function, is advancing in all the areas described above, i.e., environmental stress resistance, disease resistance, and micronutrient accumulation.  How this body of knowledge applies to woody perennials, however, is unclear.  We need better “translational genomics” – ability to apply the fundamental knowledge to crop plants, especially fruit trees. Genomic research has been identified as a research priority in the Technology Roadmap for Tree Fruit Production because of its potential to enhance crop performance, improve food quality and increase farm profits.

Genetic improvement of apple and many other fruit trees by conventional breeding methods is very slow and difficult because of heterozygosity, long generation time, and self-incompatibility.  As the research identifies and develops candidate genes for amelioration of stress injury, a technology must also be developed to introduce those genes into the tree genome.  Biotechnology can be used to some degree to overcome the obstacles to breeding, by introducing genes directly into current commercial cultivars to add the needed traits.  There is considerable market resistance to genetically engineered fruit; if the technology is to be useful, “clean” methods of genetic transformation need to be developed.  With grafted trees, the possibility exists of developing genetically engineered rootstocks with needed traits, while maintaining the non-engineered status of the scions (and the harvested fruit).  This strategy needs to be developed and its feasibility tested.

Objectives – The broad objectives of the project are to better understand the genetic basis of environmental stress and disease resistance in fruit crops and to use this information to develop genetic solutions to problems that impact the economic viability of tree fruit production.  Additionally, we are developing genetic tools that will greatly assist in addressing concerns that negatively impact the use of transgenic technologies for tree fruit improvement.

This work is being conducted through the collaborative efforts of scientists within our project and through collaborations and coordination with other projects within our facility, other USDA-ARS labs throughout the country, and public and private institutions in North America and throughout the world.

We are identifying genes associated with resistance to low temperature and drought using genomic technologies.

Transgenic technologies (silencing and overexpression) are being used to understand the function of these genes.

We are also identifying genes associated with resistance and susceptibility to fire blight, as well as conducting research to understand the function of these genes.

We are working with geneticists and breeders that have mapping populations of apple in order to determine where the genes that we identify are physically located in the apple genome and to determine whether or not they are associated with Quality Trait Loci (QTLs) for disease and stress resistance.

We are studying processes of ice nucleation and the role of antifreeze proteins in order to develop new frost protection strategies.

Flower-specific promoters are being used to specifically target “cold hardiness” genes to parts of the flower that are most susceptible to frost injury.

Transgenic rootstocks are being developed in order to assess whether or not gene expression in scions can be manipulated through genetically-modified rootstocks.  If successful, this technology will provide an approach to use transgenic approaches to solve major fruit production problems that do not result in the presence or expression of transgenes in the above ground portion of the tree or in apple fruit.

The specific research of Michael Wisniewski is highlighted below. For additional information, follow the links to the websites of the other project team members: Dr. John Norelli, Dr. Carole Bassett, and Dr. Timothy Artlip.

Michael Wisniewski Research Program Highlights:

My research focuses on:

1)                  Understanding the processes of ice nucleation and ice propagation in plants.

2)                 Understanding the genetic basis of cold tolerance in fruit trees.

Ice Nucleation and

Propagation in Plants

Factors that determine when and to what extent a plant will freeze are complex. While thermocouples have served as the main method of monitoring the freezing process in plants, infrared thermography offers distinct advantages and the use of this latter technology has provided new insights on the processes of ice nucleation and propagation. This technology is based on the fact that freezing is an exothermic event. The temperature and spatial resolution of a high-resolution infrared camera has enabled researchers to clearly define initial sites of nucleation as well as monitor the ice front as it spreads into surrounding tissues. Click on the bean leaf in the picture below to watch a video of a plant freezing.  Ice nucleation is induced by both extrinsic and intrinsic nucleators.  Ice-nucleation-active bacteria and moisture are two major extrinsic agents.  In herbaceous plants, the influence of extrinsic ice nucleators on ice nucleation can be moderated by thick cuticles or the application of synthetic hydrophobic barriers. We have demonstrated that hydrophobic kaolin can act as a barrier to ice nucleation. The situation in woody plants, however, is different.  Woody plants appear to possess native, intrinsic nucleating agents that are as active as many extrinsic agents.  The identification of the intrinsic nucleating agents in woody plants is not known.  Despite the presence of intrinsic nucleating agents, barriers exist in woody plants that inhibit growth of ice from older stems into primary, lateral appendages.  This is important because many tissues in woody plants that are frost sensitive are flowers and primary, elongating, shoot tissues that arise from buds attached to older stems.

Hydrophobic-Kaolin Treated Tomato Plants Escape Frost Injury Because Ice Initiation is Blocked

Click on the Picture Above to Watch a High-Resolution Infrared Video of a Bean Plant Freezing

Related Publications:

Extrinsic Ice Nucleation in Plants – Wisniewski, et al. (2002);  Hydrophobic Particle Film as a Barrier to Ice Nucleation – Wisniewski, et al. (2002); Antifreeze Proteins Modify the Growth of Ice –Griffith, et al. (2005);  The Effect of Water, Sugars, and Proteins on Ice Nucleation – Gusta, et al. (2004).

Genetic Basis of Cold Tolerance in Fruit Trees

Low temperature and drought are two common abiotic stresses to which plants are subjected. Together, temperature and water availability are the primary determinants of the global distribution of major vegetation biomes. Woody plants have evolved complex mechanisms of resistance and adaptation to both low temperature and water limitation involving interactions between plant anatomy, physiology and biochemistry, all of which are directly or indirectly under genetic control.

We are attempting to better understand the underlying mechanisms in cold hardiness and drought tolerance in fruit trees.  This involves the identification of genes that are specifically regulated by these environmental stresses and characterizing their patterns of expression.  It also involves understanding the function and regulation of specific proteins.  We have also used global approaches, such as proteomics and genomics, to develop a more comprehensive picture of how fruit trees respond to low temperature and water deficit.  This information will then be used to develop breeding (classic and molecular)  and management strategies that will ensure uniform, high-quality, fruit production under diverse and, at times, adverse environmental conditions.

Dehydrin Gene and Promoter Organization in Peach 

Functional Categories of Gene Expression in Apple in Response to Either Low Temperature or Water Deficit

Related Publications:

Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60 Wisniewski et al. (1999); An overview of cold hardiness in woody plants – Wisniewski, et al. (2003); Global analysis of genes regulated by low temperature and photoperiod in peach – Bassett, et al. (2006); Proteomics and low-temperature studies: Bridging the gap between gene expression and metabolism – Renaut, et al. (2003); Deacclimation and reacclimation of cold-hardy plants: Current understanding and emerging concepts – Kalberer et al. (2006); Differential regulation of two dehydrin genes in peach – Wisniewski, et al. (2006).

Biological Approaches for Managing Diseases of Temperate Fruit Crops

Background - Losses from postharvest fruit diseases range from one to 20 percent in the United States, depending on the commodity.  Therefore, reducing these losses would increase the available food supply without additional acreage or pesticide load in the environment, and reduce the need for energy, water, and capital.   Fungicides have been the major tool for controlling postharvest disease.  However, the application of fungicides to fruits after harvest to reduce decay has been increasingly curtailed by the development of pathogen resistance to many key fungicides and restrictions on their use. In particular, postharvest diseases of stone fruits caused by brown rot (Monilinia fructicola) and Rhizopus rot (Rhizopus stolonifer) are very difficult to control, even with postharvest fungicides.  In previous projects, we have developed biological control technologies for managing postharvest diseases of fruit with antagonistic bacteria and yeasts naturally occurring on fruit.  These antagonists have been commercialized.  The effectiveness and/or spectrum of activity of these antagonists, however, sometimes do not match the most effective fungicides.  Effective biological control of postharvest diseases of stone fruit and small fruit have been especially difficult because of the role of latent infections and the general lack of strong genetic resistance in commercial cultivars.  There is recognition that increased knowledge of host-parasite interactions in postharvest diseases and completely new biological approaches need to be developed  if alternative approaches are to be successful.  

Objectives - This project is designed to:  1) explore new approaches to identifying microbial antagonists that can effectively degrade melanized, fungal propagules and control latent infections, 2) determine  how the brown rot organism, Monilina fructicola, interferes or blocks innate resistance responses, and 3) block the activity of fungal polygalacturonases through the use of recombinant antibodies.

The specific research of michael Wisniewski is highlightd below.  For additional information, follow the link to the website of the other project team member: Dr. Wojciech Janisiewicz.

Michael Wisniewski Research Program Highlights

My research focuses on:

1)                  Understanding innate resistance in fruit.

2)                 Understanding the mode of action of yeast biocontrol agents.

3)                 Improving the efficacy of yeast biocontrol agents.

Understanding Innate Resistance in Fruit

The initial response of the plant to a pathogen usually involves the production of hydrogen peroxide which can function as a substrate for oxidative cross-linking of cell wall proteins and is also involved in the process of lignification. It is also functions as signaling molecule to induce hypersensitive cell death and the expression of wide array of defense-related genes in surrounding cells. In addition, the toxicity of H₂O₂ may directly inhibit pathogen growth. A compatible pathogen must either avoid triggering this oxidative response or suppress the production of reactive oxygen species (ROS), thereby preventing oxidative crosslinking of proteins, lignification and disrupting the ROS-dependent resistance of a plant. We are studying the elicitation of hydrogen peroxide and other ROS.

Related Publications:

Penicillium digitatum suppresses hydrogen peroxide in host tissues – Macarisin, et al. (2007). Control of postharvest decay of apple fruit with Candida saitoana and induction of host defenses – El Ghaouth et al. (2003). Characterization of a defensin in peach bark and fruit tissues… - Wisniewski, et al. (2003).

Understanding the Mode of Action of Yeast Biocontrol Agents

A main concern was to better understand the features of an organism that made it a good biocontrol agent. In other words, what was the mechanism of action responsible for biocontrol activity? While early studies indicated that nutrient competition and the fast growth rate of our antagonists played a major role in biocontrol activity, subsequent studies indicated a much more complex interaction between the antagonist, pathogen, and commodity. Two novel discoveries were the ability of the yeast to form a biofilm and the ability of some yeast antagonists to adhere to and parasitize pathogen hyphae. The latter report was recognized as the first reported instance of the ability of a yeast to parasitize a higher fungus. Other key factors that appeared to play a role in the efficacy of our yeast antagonists were the production of lytic enzymes by the yeast  and their ability to tolerate high levels of salts. The induction of resistance responses in the fruit by application of the antagonists within a wound or on the fruit surface was also a novel discovery.  More recently, we have used molecular approaches to examine the role of glucanases in biocontrol activity of the yeast, Candida oleophila  and to enhance biocontrol activity by overexpression of antimicrobial peptides.

Biofilm formation by Pichia guilliermondii in apple tissue (left) and on Botrytis hyphae (right).

Related Publications:

Characterization of lytic enzymes produced by Candida oleophila… - Bar-Shimon, et al. (2004). Cloning and analysis of CoEXG1, a secreted glucanase from Candida oleophila – Segal, et al. (2002). The effect of under- and over-expressed CoEXG1-encoded exoglucanase secreted by Candida oleophila on biocontrol – Yehuda, et al. (2003).

Improving the Efficacy of Yeast Biocontrol Agents

The past 15 years has seen a steady increase in the in the interest of finding alternatives to the use of synthetic fungicides for postharvest diseases control.  In particular, this has led to considerable research on the use of microbial antagonists as protective agents in much the same was as packing houses use synthetic fungicides for disease control. The success of these products, however, remains limited.  This is for several reasons, among which is the variability experienced in the efficacy of these products, and the lack of understanding of how to adapt “biological approaches” to a commercial setting.  Researches and industry have responded by trying to find “super” antagonists, which may result in the use of specific strains for different commodities, finding additives that may enhance the performance of the selected antagonists, or integrating the use of a bicontrol agent with other “physical” treatments that induce resistance in the selected commodity.  Examples of these approaches are : combining antagonists with chitosan; combining antagonists with additives such as sodium bicarbonate, calcium chloride, EDTA,  calcium propionate, etc., and, pre-treatment of prduce with UV-C radiation or heat.

Treatment of Nectarines with Hot Water Brushing Followed by Use of a Yeast Biocontrol Agent Prevents Brown Rot Decay

Related Publications:

Influence of food additives on postharvest rots of apple and peach and the efficacy of bicontrol agents. – Droby, et al. (2003); Postharvest biocontrol: New concepts and applications (2007).