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First reported in 1947, Solanum tuberosum know as late blight is the most serious disease in potato farming which was the main cause of the famines in Irish and Highland in 1845 and 1846 respectively which led to the death or emigrate of millions of people. Every year, late blight disease is the major culprit for nearly half of the losing of th totally yield of potato in developing world, vaule over 3 billion dollars.
Extensive fungicide use is the main treatment to control late blight in the developed countries now. The cost of the application of fungicides is expensive. Taking the first twelve countries joined European Union for example, the average cost of fungicide is over €300 per ha, which stands for over €400 million per year in total. In Columbia Basin, U.S., produce more than 20% of the potatoes in the country, also the most productive area in the northern America. The costs revelant to late blight mangement, mainly spent on fungicide application, on harvested potatoes represent around 38% of the potato production in the year 1995, above $30 million in the whole year. In addition, since the late 1990s, when P. infestans strains arrived in Russia, the biggest potato production country in the world, their field output of potato crops dropped by 70% in most areas. And the conditions were similar with that back in the middle 19th century in Ireland, memoried of the time of Great Famine.
Compared with fungicides, other pesticides are relative much cheaper. Taking the tomatoes production in Florida for example, not facing the threaten of late blight, the average costs for pesticides use is less than $750 per acre. Whereas, the overall value of the tomtatoes plants per acre is approximately $14,000. The cost for pest control only makes up 5% at most of the harvests' value though pesticide treatment is required weekly.While the value of corn is $322, the pesticide fees spent on the same area are $5 or only account for 1.7% of the value of corn.
In order to estimate the economic importance of late blight in potato production,
the Netherlands are taken as a case. Most data shown blew is taken from a previous report wrote by Haverkort et al. (2008). In the Netherlands, the total area cropped with potato is 165,000 ha and the average yield is 45 Mgha−1. This results in a total production of 7.9 Mt which represents an average value of
about M€ 790.
The costs of applying fungicides ars associated with of the fungicides themselves and applying them, including machines, energy, even the lost caused by machines. The total amount of fungicides applied on 165,000 ha of potato crop in the Netherlands is estimated to be 1,424 Mg. The costs of the fungicides are calculated at 61.1 million euros every year. The costs of applying on average 15 times per season (machinery, labour and fuel) are calculated at € 330 per hectare (M€ 54.4 national total). This means that costs of control (chemical + application) amoun t to M€ 115.5 for the countr y per year. Financial losses caused by incidental premature harvest represent a value of M€ 1.4. In addition, local damage incurred from Spraying machines results in a yield loss of 3%. The costs of late blight in the Netherlands at M€ 124.9 which is 15.8% of total farm gate price. Energy use and CO2 emission when growing and harvesting the crop were assumed for the additional societal costs of potato production which respersent 16% lose value 5.2 billion euros per year in the wholemarket(de Wolf and van der Kloost er 2006).
Approximately half of the potato crops lossing is attributed to unsuitable measures to control late blight and some other reducing factors. That would cost an annual losses of 23 billion euros in total. From which half of the losses, conservatively assumed, is due to late blight. The damage developed areas suffered is limited by the use of high-cost fungicides remaining 1 billion euros every year, whereas the lower yields, mainly in developing countries, sufferring from greater loss.
Drawbacks of fungicide using (from EFL)
However, the use of fungicides also led to some level of risk. Organochlorines, one of the three main pesticides, disrupt the sodium/potassium balance of the nerve fiber, forcing the nerve to transmit continuously resulting in the death of pests. Notable examples of qrganochlorines include DDT, dicofol, heptachlor and endosulfan. Organophosphate and carbamates operate through inhibiting the enzyme acetylcholinesterase, allowing acetylcholine to transfer nerve impulses to a target cell indefinitely. This causes a variety of symptoms such as weakness or paralysis, because acetylcholine is the only neurotransmitter used in the motor division of the somatic nervous system. Unfortunately, the undeveloped areas, again, sufferred more than the rest regions because of lacking protective measurements.
Montufar, located in the northern Ecuador, was chozen to be the experiment site because of the dispered rural population . Protato farmers were selected randomly, they used 38 different fungicides and 28 different insecticides. The volunteers in the control group were all capable of farming and all with similar level of age and duration of formal education. The farm team inculded the farmers who worked in the field for over foue months and family members of farmers who do not work in the field, consumers and some others directly or in directly exposed to the chemicals. The radio of volunteers to research objects is 1:2. The habits of using pesticide (e.g. Using hands to mix pesticides or no ffective protective equipments available ), presence in other fields, length of workday were recorded every day.
Relevant past health history
Number of skin conditions unlikely related to pesticides
One or more (%)
Pesticide exposure related
Number of pesticide poisonings
One or more (%)
Skin disorder (%)
Accroding to the table, the number of pesticide poisonings was always higher in the farm group (exposed and applicators). However, the consumers also accounted for part of the proportion. This indicated that even store or indirect contact of the harsh chemical elements might be harmful to hunman skin. They also found along with the use of pesticides, the odd of getting dermatitis rais 12% every year. This means if a farmer worker protect the late blight using fungicides for 10 years, the chance of him getting skin problem would be 280%.
Despite the negative effects on human and environment of pesticides, the use of pesticides also leads to the ability of pests to become resistant to the pesticidest due to the natural selection. Hence, there is now a greater than ever need for new and advanced pesticides. Resisting chemical, biological, and photolytic degradation, organic compounds that are still exiting in environment called Persistent organic pollutants (POPs). In fact, many pesticides are POPs. Along with the use of chemical pesticides, Along with the use of chemical pesticides, there will be a high possibility for POPs to get persisted in the field. Bioaccumulate in fatty tissues of animals, human included, and biomagnification in food chains lead to acute or chronic health impacts on both human especially those who exposed to the environment where pesticides are used. Main diseases caused by bioaccumulation of POPs may be irritation of the skin and eyes, affecting the nervous system, causing reproductive problems, or more seriously, cancer. POPs are also semi-volatile, enabling them to move a long distance in the atmosphere which contribute to further soil contamination. The most resistant organisms will survive the pesticide and pass the heritable mutation to their later generations. This will result in decreased sensibility to that chemical compounds land repeated failure of this series of products. In the USA, farmers lost 7% of their crops to pests in the 1940s, while the loss percentage increased to 13 during the 1980s, even though more pesticides were used.
Another problem is the progress of late blight is always fast. And the beginning stage of blight can be easily missed, and it would not affect all plants at once. Once detected, it is always too late to treat, especially in undeveloped areas. Furthermore, as mentioned before, the chemical content can be easily washed off by rain, degrated by sunshine (high temperature) or evaporated. Therefore, to protecct the harvests, overdosed fungicide sparying is usually the only way farmers are able to perform. According to Kromann_et_al_2009, some experiment performed in Ecuador had provided a reasonable minimum use of fungicide spary within the shortest period. They chose mancozeb and propineb the most common used in this region as the fungicide. And the influence of rain was treated as the main factor because of the local weather condition of the Andean which is humid but cool.
Hence, a late-blight free potato would especially impact people's food security and income in developing countries where losses would become much smaller and where the a creage of the potato crop would increase even more rapidly than it already does at present. Moreover, worldwide, use efficiencies of land, water, nutrients and energy would greatly improve when yields would no longer be reduced by late blight.
What caused late blight
pathogenic oomycetes are blame for several devastating diseases in plant crops, including the most serious ones. The spores of these oomycetes can survive in effected potato tubers or soil in the environment over winter and are well known for their abilities to spread rapidly under the warm and wet weather. Especially those spores produced on the leaves of infected plants, in terms of potatoes and tomtatoes, can be brought into the air, travel hundreds of miles at most and land on other hosts, when the conditions of temperatures and humidity are sufficiently for a couple of days, normally above 10 â„ƒ and 75% respectively. Even worse, rain and irrigation can wash spores into the soil to cause new infection. This may rot the tubers before or after harvest. And if the producers or the consumers saved some for next year to plant or just simply missed one in the field, they could be in trouble again.
At least two mating types are required to ensure the life cycle of the spores, because the asexual zoospores can only survive in host tissues between seasons(Merry et al, 1997). A-1 was the only mating type found in almost all the infections worldwide before the year 1976. With the production of numerous zoospores, this blight can completely destory an extensive area of potatoes within few days. After the year 1976, A-2 was shipped from Mexico with tons of potatoes to Europe and then spread and established to the whole world. Nowadays, Phytophthora infestans are able to produce zoopores that resistant to the outside rivalry environment to survive years. Furthermore, the type 2 blight has strengthened their pathogenicity factors and enhanced resistance to most currently used fungicides.
Microbe-associated molecular pattern (MAMP) are microbes that can active the defence response of plants immune system. When combinated with a pathogen receptor binding virulence signal, it becomes Pathogen Associated Molecular Pattern (PAMP). Thses two kinds of molecules are detected by plany immunology continuously. The highly evolutional conserved motifs of PAMO can be recognized by pattern recognition receptors of the innate immunr system. Once fungal pathogens force the host to release cell wall molecules, PAMPs termed Damage-Associated Molecular Patterns (DAMPs) that active the defence resopnse.
Fungal effectors fall into two groups: those that are secreted into the host apoplast, and those that are translocated into the host cells (De Wit et al., 2009§). Infection involves two phases: a biotrophic phase up to 36 h post inoculation in which P. infestans forms haustoria and requires living plant tissue, and an ensuing necrotrophic phase in which infected host tissue becomes necrotic.
While the mechanism of translocation in either fungal pathogens or oomycetes is unclear, a conserved motif, RXLR-EER motifs, has been identified as being sufficient for effector uptake by host cells (Whisson et al., 2007§). Pathogen AVR3a comes from the potato late blight pathogenP. Infestans, like other AVR proteins, has a signal peptide for secretion from the pathogen, followed by the motif RXLR (where X is any amino acid) and an acidic region, ending in the sequenve EER. While RXLR-EER is similar to another signal response for effector delivery in animal cells not only in sequence but also in location. In addition, a sufficient flanking sequence is also needed for the host-drived translocation (Whisson et al., 2007§). And they are found to be functionally redundant that all the candiate genes expressed can easily help oomycetes to change the composition to escape the detection. In short, many of these cytoplasmic oomycete effectors consist of an N-terminal region involved in secretion and translocation, as well as a C-terminal domain response for host defence (Morgan and Kamoun, 2007§). In recent years, a signature motif at the N-terminus has been identified and characterized as a critical component that not only guides oomycete effector identification but is also a critical component in the function of these secreted proteins during host interactions (Whisson et al., 2007§).
Resistance protein R3a recognizes Avr3a in the host cytoplasm, triggering the hypersensitive response. Secretion of Avr3a as a fusion protein by the Type three secretion system (T3SS) which used by bacterial to transfer their effectors into host cells was demonstrated by coinfiltration of Nicotiana benthamiana leaves with Agrobacterium tumefaciens that delivered the R3a gene, and yielded a clear hypersensitive response. In contrast, a hypersensitive response was not observed when Avr3a was fused to a bacterial nonsecreted protein or a type II secreted protein (Supplementary Fig. 3§), or when the T3SS was disabled, indicating that translocation was dependent on the T3SS (Fig. 1§). These results confirm that Avr3a must be delivered inside the plant cell for recognition by R3a, implying that Avr3a translocation by P. infestans may require a structure and environment-as is created by the haustorium and extrahaustorial matrix-and/or a mechanism functionally analogous to the T3SS.
Two main types of potato resistance to late blight disease have been distinguished: field resistance and true resistance (R gene mediated resistance). Although field resistance shows a more durable resistance to late blight, R gene mediated resistance is much easier to operate (Ross, 1986). True resistance, virtue of hypersenstivity, is based on the detecte, isolate and clone natural existent genes, normally from wild Mexican potato, known as Solanum demissum. It is the most widely used and the most efficient way to treat potato late blight.
PR genes, a small part belong to the large defensesignaling network confer resistance to many pathogens. I will briefly introduce the mechnism of R gene mediated responses in the following paragraphs.
R gene mediated responses.
Each R gene confers resistance to a specific pathogen. Hypersensitive response (HR) is always the first phenotype appears in the plant defence system when respond to R gene mediated resistance. Programmed cell death (PCD) is induced by HR then, occurs at the site of infection and reveals lesions that protects virus or infections from spreading to healthy cells nearby immediately. Systemic acquired resistance (SAR) occurs in the distant tissues following the railar response, hypersensitive response, is treated as the second phenotype of this resistance. It is evolutionarily conserved that inhance the immunity of those tissues to the infection or the similar ones. Once initiated by the accumulation of endogenous salicylic acid (SA) (essential condition), it can last for weeks. This response is related to the induction of several genes called PR (pathogenesis-related genes).
SA and jasmonic acid (JA) are believed to play an important role in the complex signaling networks that mediate plant denfence response to invasion. In response to pathogen infection, SA is synthesized via the isochorismate pathway (Wildermuth et al., 2001§). Signaling proceeds via the central regulator NPR1, which oligomerizes in the cytoplasm, facilitated by Snitrosylation (Tada et al., 2008§). Thioredoxin catalyzes the SA induced release of NPR1 monomers, which translocate to the nucleus where they interact with TGA transcription factors and induce expression of specific defense genes (Dong, 2004§; Pieterse and Van Loon, 2004§; Tada et al., 2008§). Additional components, such as the transcription factor WRKY70, are required for full expression of SAresponsive defense genes
R protein structure.
Strictly speaking, most resistence genes discovered so far belong to the NBARCLRR family of plant R genes. There are three motifs in the nucleotide-binding site that required in ATP/GTP binding proteins. The nucleotidebinding (NB) site domain and neighbouring R gene sequences are named as the NBARC domains because they are similar to the equal regions of Apaf1 and CED4, the death genes of metazoan cell. With the help of other associated domains, which come from pathogenic bacteria, leucine-rich repeat (LRR) can represent protein-protein interactions. The LRRs of mammalian Toll-like receptors (TLRs) interact with pathogen-derived molecules to start defence responses. Each R protein resistent to different pathogens or viruses, despite their similarity in sequences or structures.
R protein domain function.
Three domains of R genes all play necessary roles in pathogen recognition and/or signalling by using mutations in N protein of Nicotiana glutinosa.
There have also been attempts to identify the domains that confer recognition specificity on R proteins. Domain swaps between different alleles of the flax L gene, which confers resistance to a fungal pathogen, indicated that both the LRR and the TIR domains have roles in the recognition of R proteins. Several residues in these proteins might be undergoing positive selection, and most of them are found within the LRR domain (J.L.M.S. and S.P.D.K., unpublished).
Intramolecular rearrangements within R proteins have also been shown to be involved in recognition. Coexpression of CCNBARC and LRR domains or CC and NBARCLRR domains of S. tuberosum Rx1 results in a HR in the presence of the PVX coatprotein Avr (avirulence) determinant, mimicking the function of the intact Rx1 protein. The CCNBARC and LRR or the CC and NBARCLRR coimmunoprecipitate, but this interaction is abrogated in the presence of the coat protein. These results indicate that there are specific interactions between the domains of S. tuberosum Rx1 and that the coat protein disturbs the native conformation of Rx1. It is probable that molecular interactions within S. tuberosum Rx1 hold the protein in a conformation that is poised for signalling but is inhibited from doing so. Addition of the Avr determinant releases the inhibition and allows defence signalling. Evidence for intramolecular interactions has also been obtained for the tomato Mi1 R protein, which confers resistance to a nematode.
Recognition of Avr determinants.
Any protein component of a virus can function as the specific Avr determinant to elicit resistance mediated by a given R gene (Table 1). Despite the availability of several cloned R genes and their cognate Avr determinants (Table 1), progress in understanding how pathogen Avr proteins are recognized has been slow. Initially, receptor-ligand models were proposed to describe the interactions between R and Avr proteins (Fig. 2a). However, this simple model does not apply to any viral R-Avr pair examined to date, although it has been shown to apply in two cases of bacterial and one case of fungal resistance2. A more sophisticated model of R-Avr interactions invokes the involvement of Rproteincontaining complexes. The 'guard hypothesis', originally proposed by Van der Biezen and Jones19, postulates that R proteins (guards) are constitutively associated with host cellular proteins (guardees) that are required by pathogens for infection (Fig. 2b). On infection, the pathogen causes modifications to the guardee that are detected by the guard (Fig. 2c). Any protein modification that can alter the quaternary structure of the guardee could result in detection of the pathogen. This activates the guard to initiate a signalling cascade that culminates in the resistance response.
Four different lines were chosed to perform this experiment. Foliar resistance, tuber resistance and yield performancce were evaluated respectively. All of the RB transgenic lines exhibited an increase in resistance to P. infestans when compared with their non-transgenic counterparts. The increase in foliar resistance did not significantly de-crease tuber yield, the most important aspect of potato production value, in the absence of late blight. We also were unable to detect any significant effect on yield after the incorporation of the RB gene into various potato cultivars.
RB-transgenic tuber resistance assays
Assay of foliar resistance to P. i n -festans
To achieve durable resistance against the late blight, there are eight basic principles:
1. Bacterial vectors are used to transform target genes into plants to contain the same phenotypes as the origin. The resulting resistant potato varieties contain potato genes only without any bacterial genes and no new varieties are created. The start and end products are potato varieties made up of potato genes only.
It is better to use natural R genes from the wild potato species because it was assumed that this approach was ethically more acceptable to the public than the use of alien genes from not crossable species.
3. Due to the possibility of a single R gene being rapidly broken down, it is better to insert several resistence genes, normally two, four at most, from different species to improve the duration of resistence. In addition, Phytophthora infestans have the abbility to bypass the R genes. Hence, it is not wise to stack R genes at the same site. Instead, decorating those different varieties in different locations at different time.
4. DuRPh does not use selection markers such as herbicide tolerance or antibiotic resistance to select for insertion of the desired gene(s) in the variety. The achieved late blight resistanc e is the main marker so the variety is marker free. To assure that the R gene is (or the R genes are) present the genotypes that appear resistant to all known pathotypes of Phytopht hora infestans are tested using PCR techniques.
7. DuRPh dedicates a considerable proportion of its efforts to communication with all stakeholders concerned. By being transparent regarding philosophy, approaches, techniques, and results it is up to individuals and groups to make their own decision about appreciation of the techniques used.
The exploitation of the R genes with proven efficacy and known not to be homologous to previously discovered ones is by protecting the intellectual proper ty and making them available (not exclusively) to private potato breeding companie s; once these genes are exploited in new varieties the prvate companies reimburse the owners through breeding right agreements. For developing countries where food security is an issue human itarianaid through R genes is an option.
PR genes are categorized into more than fifteen families (Van Loon et al., 2006§), PR1 family, the most commonly used disease resistance markers, included. The PR1 family of genes are extensive, highly conserved and represente in neraly all plants, with homologs present in fungi, insects and vertebrates (Van Loon et al., 2006§). However only a single member of the PR1 family is actived by pathogen infection, insect feeding, or chemical treatment(Van Loon et al., 2006§). In contrast to the PR1 family, several additional PR groups have also been widely studied, including members of the PR12 family, also known as defensins, which have members exhibiting antifungal activity.
Resistance is termed durable if it continues to be effective over multiple years of widespread use, but it somtimes appears limited value baceuse invaders may evolve quickly to overcome this resistance. However, it is very time-consuming (only the analysis step may take several months) use recent maping based technology to discovery new R genes. Hence, it is importance to find a new method to cut the time of identifying advenced R genes