Báo cáo Nghiên cứu khoa học Tristeza disease and its management

TRISTEZA DISEASE AND ITS MANAGEMENT NGUYEN VAN HOA SOUTHERN FRUIT RES. INST. What is virus? It is only the DNA or RNA fragment, it may be single or double strains with the coat protein. DNA or RNA may be free from coat protein and self replication using the genetic material from the host (virus infected tree). It can move from one cell to another through plasmodemata and become systemic infection through phloem tissues and the whole tree got infection. Potential virus diseases on fruit crops and its management Introduction  Tristeza virus was considered original from China. Tristeza was called as quick decline in America, is the most destructive disease and spread through out the world.  Only 2 decades after tristeza was introduced from Africa to South America in 1920s, the disease caused much damaged to citrus industry in Argentina, Brazil and Uruguay. Especially on sweet orange grafted on sour orange. Virus normally does not express the symptoms on the resistant combination between rootstock and scion of mandarin and Trifoliata orange. Symptoms of CTV on different citrus species/clone  Citrus tristeza virus (CTV) caused many different symptoms based on the virus isolates, Citrus variety and the combination of rootstock and scion.  In Asia, many isolates/strains such as seedling yellows (CTV-SY), tristeza (CTV-T), stem pitting - (CTV-SP), and one mild strain. Any strain can get infection on one tree or infected by many strains at the same time. Quick decline, or tristeza disease The following varieties got infection with quick decline if it was grafted on sour orange, pumelo or rough lemon: Sweet orange, mandarine (including Satsuma and Ponkan group), Tankan, Iyo, Tangor, varieties belonging to tangelo, and grapefruit. The tree got infection and shown yellowing and wilt and then die within years. If it is grafted on resistant rootstock such as Trifoliate or mandarin, it will be recovered soon. Symptoms  Seedling yellows Young seedlings of sour orange, Natsudaidai, rough lemon infected with CTV-SY strain, the canopy become yellow and die. If it is grafted in the resistant rootstock it will be recovered quickly.  Stem pitting disease All most the citrus cultivars infect with the stem pitting strain, even when it was grafted on resistant rootstock. Grapefruit, tangelo, tangor, sweet orange, Iyo, Yuzu and natsudaidai are very sensitive to the disease, even the Satsuma and Ponkan are resistance. The susceptible variety infect show stem pitting on trunk, branches, twigs. Infected tree become dwaft, poor development and sometime even died. Lead the trees have more flowers but fruit setting less, fruit becomes small, deform. Some varieties show brown spots on fruit or fruit with brown spots and gum oozing. Symptoms  Citrus tristeza virus (CTV) is the causal agent of quick decline on citrus grafted on sour orange.  At first, the infected tree bear small leaves, death of young and small twigs, bearing small fruits.  In some cases, the infected tree got fast wilting and death within weeks.  CTV caused incompatibility between rootstock and scion leading to the girdling around the trunk base with the yellowing symptoms.  These trees shown stem pitting on the xylem tissues when the bark removed. Vein clearing due to CTV Tristeza disease on citrus Virus particle under microscopy Quick decline due to CTV infection Stem pitting symptoms on the xylem tissues of citrus Symptoms (cont.) Sweet orange cv. Valencia grafted on sour orange rootstock affected by Citrus tristeza closterovirus (CTV). Mexican lime seedlings (Citrus aurantiifolia). Left: healthy; right: inoculated with CTV. Dieback of sweet orange grafted on sour orange caused by CTV. Symptoms (cont.) Inverse pitting, honey combing, or pinholing induced by CTV below the bud union on sweet orange trees grafted on sour orange rootstock. Stem pitting induced by CTV on small branches of grapefruit grafted on the tristeza-tolerant rootstock Citrus limonia. Stem pitting induced by CTV on a grapefruit trunk. Left: grapefruit from a healthy tree; right: four grapefruits from a tree grafted on sour orange and infected with CTV. Vein clearing induced by CTV in inoculated Mexican lime (C. aurantiifolia) seedlings incubated in a glasshouse at 18-25°C. Symptoms (cont.) Symptoms of stem pitting on xylem tissue and yellowing on fruit Causal organism  Causal organism is a virus with filamentous rod shape, length of 2,000 nm and 15 nm width.  Virons stay in the phloem tissues of the host and interfere the nutrition and water uptake in the tree system.  The optimum temperature for virus infection and multiplication is 20°C - 25°C. Causal organism: Closterovirus Virions have a simple construction and consist of a capsid. Virions are not enveloped. Capsid is elongated and exhibits helical symmetry. The capsid is filamentous, flexuous with a length of 2000 nm and width of 12 nm. CTV strains  Citrus tristeza virus has many strains cause different levels of damage.  Mild strain spread fast through aphid and infected planting material. These strain do not cause quick decline of sweet orange grafted on sour orange.  Quick decline strain occur and cause much damage. When it is grafted on sour orange causing dwaft of the tree. However, when it was grafted on resistant rootstock, but showing symptomless event when it gotinfection. Symptoms due to deferent CTV strains  In some countries, CTV strain can cause stem pitting on any kinds of rootstock.  The stem pitting showing on the xylem tissues of trunk, branches and twigs. Looking from outside, the branches become twisting and bristle. The tree become less growth, yield, bear small fruits. Detection  Normally, all the strains can be detect through indicators tree as Mexicane lime.  ELISA can be use with the polyantisera. One monoantibody named MCA-13, can be used for ELISA, and detect all the strain except mild strain. Beed ELISA showing enzyme mediated colour reaction. Microplate showing Double antibody sandwich-Indirect ELISA results of enzyme mediated reaction of healthy and CTV infected samples. B and G rows - Buffer control C and E rows - Healthy sample D and F rows - Infected sample A and H – Empty wells I I H H Screening of citrus isolates against CTV by DIBA From Top No.1 to 14 – Glass house mantained Puna isolates 1st row - CTV +Ve No. 15 to 28 - Glass house mantained Bangalore isolates 2nd row – Healthy No. 29 to 42 - Glass house mantained New Delhi isolates No. 54- Healthy No. 43 to 47 - Glass house mantained viroid infected plants No. 55-56 - CTV +Ve No.48 to53 – Field isolates Electron microscopy showing Decoration of CTV particles by homologous antiserum Disease transmission  Virus Tristeza is transmissible through aphid in a semi-persistent manner. Aphid can acquire the virus within minutes when it feed on infected tree and then transmit the virus to the healthy tree within a minutes when it feed on the healthy one.  Aphid lose their ability of transmission within 24-48 hrs after leaf the infected tree. Toxoptera citricida is the most effective aphid in transmission. Aphis gossypii of litchi or cucubitae, Aphis spiraecola and Toxoptera aurantii are also transmissible aphids. Myzus persicae – vector transmit Tristeza virus (mild strain) Aphis citricola – Cũng là tác nhân truyền bệnh Tristeza Toxoptera citricidus – vector transmits sever citrus tristeza virus strain. Control measures  When the tree infected with serve strain, there is no way of cure, so that do not multiply from this tree. When on the orchard, there is some randomly infected trees, these trees should be cut off and replant with the free disease seedlings, which have been grafted on resistant rootstock. Even though the aphid can transmit the virus very fast, the chemical or biological control can not reduce the infection speed. The transmission speed is too fast, it occur before the control method is conducted. However, the control method carried out in the nursery and from the source of scion is very effective. Control measures (cont.)  The combination of certified rootstock- scion should be used and the resistant rootstocks such as trifoliate orange, Sunki and Shiikuwasha (C. depressa Hayata) should be used. Some of the hybrids as Troyer citrange or Swingle citromelo, are potential rootstock for resistance to tristeza.  The free-desased seedlings through STG and heat treatment should be used. Control measures (cont.)  Cross protection, the method of inoculation of the mild strain to control the infection of the serve strain, this method has been widely used in South Africa, Australia to fight against quick decline strain and in Brazil to control stem pitting strain. CROSS-PROTECTION Plants systemically infected with one strain of a virus, will not develop additional symptoms when inoculated with a second strain of the same virus. This phenomenon forms the basis of cross-protection tests. This phenomenon is well known with NEPO viruses. Inhibition occurs between closely related strains and generally not between unrelated strains or viruses. The mechanism of cross protection There are many strategies to explain the mechanism of cross- protection General theory (A) Replication site competision. Diferent strains of the same virus : they have the same site for replication, which ahs been occupied by the mild strain so that another strain can not multiply (B) Lack of the material which is necessary for the metabolism. (C) Help the tree produce resistant substance which can help the tree resistance to the virus. Theory base on the activities of the coat protein of the VIRUS (A) ‘The absorption' of the infected material to the infected cell: The virus particles adhesive together when it infect: TMV (B) The sticky of the coat protein into RNA sequence of the virus: Before replication, the coat protein must be remove, just when it is removed, the coat protein already there bite to the RNA so that the replication process can not be done. CROSS-PROTECTION can not be recommended as a general practice because: 1. Mild strains also reduced yield upto 5-10%. 2. The immunised crop may act reservoir of virus from where more sensitive spp. or varieties can be infected. 3. The mild strain may turn up as sever by mutation. 4. Sever disease may appear with mixed infection of mild strain and unrelated viruses. Genetic engineering • The resistant gene from the virus CTV: Coat protein gene, replication gene, etc. have been used for transformation