INTERNSHIP REPORT

1-3-2009 to 30-5-2009

PLANT VIROLOGY SECTION AARI, FAISALABAD

Submitted to: Mr.Inam-ul-Haq (Plant Virologist) Submitted by: NAYYARA IMTIAZ 2005-ag-1632 B.S.c (Hons) Horticulture

INSTITUTE OF HORTICULTURE SCIENCE UNIVERSITY OF AGRICULTURE FAISALABAD

ACKNOWLEDGEMENT
In the name of ALLAH the beneficent the Merciful whose blessings are cherished fruit to my efforts to overcome the problems and difficulties. So all thanks to Him. I m thankful to all of the members of Institute of Horticulture Sciences specially Dr. Aslam pervez (Director) and Dr. M.Jaffar jaskani (Supervisor) For their kind attitude during my internship and whole degree programme. I would like to take this opportunity of thanking to all of the staff of Plant Virology Section of Ayub Agriculture Research Institute in general and to my Supervisor Mr. INAM UL HAQ in particular for their sympathetic attitude, Kind guidance and supportive behavior during my internship.I would also pay my thanks to MISS. ASSIA BATOOL for her supportive behavior. In the end I will pay my thanks to my loving parents, brother and sisters for their love, good wishes, endless prayers, and supportive behavior. Nayyara Imtiaz.

To Whom It May Concern

It is certified that Miss NAYYARA IMTIAZ D/O IMTIAZ ALI, student of B.Sc. (Hons.) Horticulture with Reg. # 2005-ag-1632 has completed her Internship training programe in Plant virology Section, Ayub agriculture research institute Faisalabad from March 1st, 2009 to May 30, 2009. I found her very honest, hardworking, regular and obedient person. She did her job with great interest. I wish her every success in her life and a prosperous future.

----------------------------------------INAM UL HAQ (PLANT VIROLOGIST)

Internship supervisor

--------------------------------------Miss. ASIA BATOOL (ARO) Co supervisor

Contents
Lectures and discussion
Introduction Introduction and importance of potato Seed potato production technology Diseases of potato Insect pests of potato IPM for potato Diseases of general fruits and vegetables Production technology of Citrus Production technology of Mango

Visits
Vegetable Research Area

Activities
Virus free seed potato production through TISSUE CULTURE MS media preparation Inoculation in the test tube Monitoring plant growth in the test tubes

 Shifting and monitoring growth of plants in the sand trays Shifting and monitoring plant growth in pots Monitoring and maintaining plants in the tunnels ELISA for watermelon mosaic virus

Ayub Agriculture Research Institute

Agriculture bears great economic importance. it plays fundamental role in strengthening national economy of a country benefit of other natural resources. radically Pakistan as an agricultural realm: its fecundity is above board. agriculture has been the single largest sector playing applaudible role in earning remarkable foreign exchange. this role can be further extended by improving the existing farming system .in most of the far flung and rural climate our farmers are still sticking to the absolute and traditional farming system that result in poor production and return. this is the age of science and technology therefore, our agriculture needs to be attuned and re-oriented according to modern inventions. our agricultural scientists are intensively engaged to ransack new technologies and methodologies to put agriculture on the right track leading towards prosperity and self sufficiency. Ayub Agriculture Institute(AARI) was established in 1964.mainly,Ayub department is consisting of 4 major wings. out of these, Agriculture Research is one of the most important one, which governed, administer and is responsible for the research within the Punjab province to caster its need. Ayub Agriculture Research Institute is the premier organization of Agriculture department and is located at Faisalabad ,a beautiful city located about 150

kilometer from Lahore-the capital of Punjab Province. main objective of AARI is to conduct research to enhance agricultural productivity and focus on the research leading to resolve the problems of growers. Ayub Agriculture Research Institute serves the need of entire Punjab Province for the improvement in all crops grown in the province such as sugarcane, oil seeds, vegetables , Horticultural and other crops. research at AARI provides huge benefits of poverty alleviation through productivity improvement and enhancement integrated with Plant Protection and management practices. A ARI meets this challenge through applied research, day to day problems of the growers, human resources and development and dissemination of information. the results of the research are transferred to different regional research institutions spread through out of the province for further testing and improvement under different ecological zones. many laboratories have been upgraded at AARI, which are contributing significantly in their field of specialization.

PLANT VIROLOGY SECTION

Introduction
plant viruses are one of the major agents that are causing great losses to vegetables , field crops and fruit plants. these losses are even more pronounced in vagetatively propagated crops where pathogen passes through one field generation to the next .Plant Virology Section was established at Ayub Agricultural Research Institute ,Faisalabad during 1973 to study various aspects of viral diseases of crops, potato in particular and develop strategy for the control and management of these diseases. high quality disease free potato seed is a single one most important factor in potato production but local production

of certified seed is not enough to meet the requirements of the growers, which force the country to import potato seed involving huge amount to foreign exchange. in view of the situation Punjab Government added a small tissue culture unit in Plant Virology Section in 1984.

Production of high quality seed potato was started using tissue culture technique which ensures virus free seed stock .Now the section is supplying pre basic seed potato to seed Companies and progressive growers for continuous and reliable feed back for seed production cycle.

OBJECTIVES
1:

Survey, identification and study of plant viruses.

2: Screening of plant material to find resistant sources against viruses and facilitate breeders for evolving resistant varieties. 3: Control of viruses by thermotherapy / chemotherapy. 4: elimination of potato viruses through tissue culture and micro propagation of healthy plant material

PLANT TISSUE CULTURE

Plant tissue culture (PTC) techniques are used for growing plants in a sterile controlled environment for the purpose of mass production, germ plasm preservation, plant breeding, physiological studies, and genetic engineering. By using plant hormones and other growth regulators, small plant parts can be induced to produce hundreds of small "plant lets", which can later be grown in a greenhouse, in the field, or as house plants. In plant tissue culture experiments, we use ex plant (pieces of differentiated tissues)to initiate their growth in culture. When an ex plant from differentiated tissue is used for culture on a nutrition medium, the non-dividing quiescent cells first undergo certain changes to achieve a meristematic state. The phenomena of reversion of mature cells to the meristematic state leading to the formation of callus is called dedifferentiation. The component cells of callus have the ability to form a whole plant, a phenomena described as re differentiation. These two phenomena are responsible for regeneration of whole plant from a small piece of tissues. Plants are totipotent that is plants are capable of regenerating a complete plant from a single cell but an animal cell losses this property. This property forms the basis for tissue culture in plants only. In the 1980s by the exciting prospect of deliberately and specifically modifying the characteristics of a plant by genetic manipulation. The description of the Development of plant tissue culture as a technique in its own right was augmented mechanism of infection of plants by the soil organism Agro bacterium tumefaciens was followed by its modification to deliver foreign genes. The first demonstrations of stable plant transformation were then made by both American and European scientists (Horsch et al., 1984, Zambryski et

al.. 1983). Development of standardized methods of plant transformation followed, using A. izitnefizciens or Agrohacterium rhizogenes, or direct methods like microinjection, electroporation or particle bombardment. Methods were suitable first for herbaceous dicot plants like tobacco (Nicotiana lahacum), petunia (Petunia hybrida) and tomato (Lycopersicon esculenlum) (e.g. Horsch et a!., 1985), later for woody dicots and lastly, rnonocots (e.g. Gasser and Fraley. 1989). It has already been indicated that the techniques of tissue culture are essential tools for plant genetic modification. The techniques of plant tissue culture are therefore no longer limited to the specialist tissue culture laboratory but are also the everyday tools of the plant molecular biologist. Materials in routine culture now include all the major food and fiber crops as well as many other species of economic importance, including both angiosperm and gymnosperm trees. Arabidopsis phaliana (arabidopsis), the subject of the first plant genome project to be completed and a model species in plant science, is also widely used in cell and tissue cultures. Techniques of plant tissue culture have, therefore, been developed over more than half a century and include many species. Often, published protocols have been presented without a clear rationale for their use, or without attempting to explain why modifications were made for a particular species.

History
In the beginning of plant tissue culture was made as early as 1898* when a German botanist. G . herbalist successfully cultured fully differentiated individual plant cells; isolated from tissues in several plant species. For about thirty five year ( i.e up to 1934) little further progress in cell culture research was made ; although culture of embryos, root and other tissues was achieved in this period .during 1934-1939; due to discovery of the importance of auxins and

b- vitamins the foundation of plant tissue culture was laid down by three scientists (Gautheret;White and Nobecourt) ; even though only small pieces of tissue and not individual differentiated cell could be grown in cultures. During the next twenty years (1940-60); a variety of chemical were identified for their effect on cell division; growth and differentiation. Thus; by early nineteen sixties; the methods of in vitro culture of plant cell; tissues and organs was reasonably well developed.

Applications
1-In vitro preservation of germ plasm
A low-maintenance in vitro germ plasm collection provides a costeffective alternative to plant under field conditions and diseases; and also provides a method for conservation of species that are difficult may also eradicate pathogens from imported germ plasm. Rapid multiplication of superior clones and maintenance of uniformity is also possible by tissue culture.

2- Elimination of systemic pathogens and rapid multiplication of disease-free clones

A prerequisite for the use of micro propagation for germ plasm storage and exchange, and for commercial large-scale plantings, is the use of diseasefree plants including heat treatment, meristem and callus culture, chemical treatment, and a combination of these treatments. On the other hand, meristem or callus, or a combination of heat treatment and meristem culture, have been used to eradicate systemic diseases of sugarcane plants such as SCMV ,Fiji disease fijivirus (Wagih et al.1995) and maize streak geminivirus(Roth 1969) .In contrast, neither meristem culture nor heat treatment has been successful in eradicating sugarcane bacilli form badnavirus from

sugarcane free plants can be produced by regenerating the plants from cultured tissues derived from 1-virus free plants 2-meristems which are generally free of infection 3- meristems treated with heat shocks(34-36 C)to inactivate the virus 4- callus which is usually virus free like meristems. Further in callus culture virus infection can be completely eliminated through repeated subculturing.

3-Transgenic Plants
In vitro culture of a wide of range of commercial plant cultivars is essential in developing reliable for gene transfer for cultivars improvement. The establishment of embryogeneic callus and regeneration of transformed plants are required for genetic transformation studies using micro projectile boom bombardment.

Introduction and Uses of Potato

Potato was probably first cultivated more than 800 years ago in Aden Holland (Titicana) Grown today in more than 125 countries around the world. Is available in over 5000 cultivars from more than 10 wild types. Provided with its tiny starch grains. Water content is up to 80%. It was the first vegetable to grow in space.(on bore space shuttle Columbia 1995) Rich in vitamin C and essential minerals. Anti oxidant (prevent aging)

 Does not absorb salt during cooking. Used to make glue. Used as fluid.

World wide Ranking

Rice. Wheat. Maize. Potato

Uses
Starch that is impressively versatile commodity. Paper and card board manufacture. Biotechnology raw material. Provides raw material/substrate for media preparation.

Textile industry(starch and ether give abrasion, resistance and smoothness.

Pastes and Glue.

 Pharmaceutical and Cosmetics. Used in Powders, toothpaste and creams.(modified starches and additives in tablets and help them to dissolve it in body. Detergents. Biologically degradable materials.

Exotic potato varities

Arinda. Banba Carnaval Orla Spirit Rubis Kufri badshah Courage Laddy rosetta Hermes These varities are tested but not certified.

Local potato varities

Old: Cardinal, Desiree and diamond. New:SF-5, FD8-1, FD1-10.

SEED POTATO PRODUCTION TECHNOLOGY

There is difference between seed potato and common potato due to the characteristics of seed.The seed potato is of definite size that ranges between 35mm-55mm and the proper size of potato is just like the egg size which is ideal for potato.The quality seed of potato should be free from any lesions or spots of diseases like black scurf ,common scab and powdery scab.The seed tuber should be free from rot either soft or dry rot.It should have eyes for sprouting.There should not be any wrinkle and it should have sprouts before sowing.Sprouts should not be too long and surface should not be flaxed.So the quality seed is the prerequisite for growing maximum seed yield.

IMPORTANCE OF POTATO
It is one of the most important vegetable which is more nutritious and it is mostly used as essential diatery item in dishes.In developing countries,it have great importance to overcome the food deficiency.It is called cheep food i.e available through out the year.So far as the importance of production factors such as soil preparation,seed rate,varities,time of sowing,fertilizer,irrigation,earthing and other important pest and disease control and timely cultivation the quality seed has the great role getting optimum potato yield.Potato seed is costly input.The cost of the seed is up to 40 % of the total expendure.So the seed has got prime importance.It has been observed that the potato planted in spring season is prone to sucking pests particularly the Aphid which plays great role in transmission of viral diseases. Two types of aphids,Myzus persicae and Aphis gossypii are important.Therefor the spring potato crop should not be sown for seed production due to the transfer of viral diseases, quality of seed potato is reduced due to infection of viruses as the population of these insects in spring season is high. If the seed is healthy and

planted in spring season 15-20%virus infection is likely to occur. Due to

the use of effected seed, not only the yield and quality of potato is reduced but seed degeneration. So due to high attack of aphid on spring potato crop, spring cultivation of potato is discouraged specially for seed production.The optimum period for healthy seed potato production is
autumn season when activity of sucking pest, particularly the aphid is less. Before few years, the seed potato was imported from foreign countries like Holland and the seed reached in the end of December or in the early January. So the imported seed had to be planted in spring season. After long experiments it was observed that seed cultivation in spring season is hazardous and so much so the expenditure on import of seed potato are also high.So the strategy was revised to avoid the spring cultivation and a plan for autumn sowing was prepared to get rid of aphid infestation and transmission of virus diseases.

Steps for potato cultivation

Steps before cultivation: Quality seed of approved varities like cardinal, desiree and diamond should be preferred over the other local varities. There are so many exotic varities available in the market and floating for business without proper certification and registration by Federal seed certification department. Seed potato should be free from dry rot or soft rot and cracking. They should not be used as seed but should be buried deep in soil. Select fertile and such soil where potato has not been cultivated for 2-3 years. The seed should be taken out from cold store 7-10 days either of sowing and should be placed under shady and airy space. So that the sprouts may

emerge. The seed potato should not cut as it gets easily infected with soil born fungi, bacteria and nematodes.

Steps During cultivation:

Seed rate should be 12-15 bags of 100kg/acre. If varity is not of standard than increase the seed rate to select only the healthy plants. Plant in autumn season. Never sow in spring for seed potato. The potato should be sown preferably in early October. Some farmers cultivate potato in mid-last weak of September during which temperature is high and the seed tubers are rotted.This results in low plant population and consequently low potato yield. It means that the potato cultivation should not be too early or too late as there is likely hood of attack of insect pest and diseases. A balanced dose of fertilizer,100 kg N 50 kg P and 50 kg K is the economical dose as the fertilizer costs are increasing so low input of fertilizer should be used. All the quantity of P and and K and half of N should be applied after sowing.In rice field where potato is to be sown 5 kg ZnSo4 35% should also be used. Before cultivation of potato FYM within 20-25 tones/hac shoul be added in the soil before 1-2 months.It should not be raw as the half rotten or raw FYM will increase the incidence of soil born diseases particularly the scab. Planting geometry*R distance should be 28 inches i.e 70 cm.p*p should be 6 inches i.e 15 cm.

 Planting depth should be 4-6 inches i.e 10-15 cm because we have to earth up the soil to provide support, avoid greening and to increase the number of stolons. Steps After cultivation: The day potato is planted it must be irrigated just after the sowing. It is the first irrigation and should not be delayed otherwise there are chances of rottening and late emergence. Next irrigation should be at 5-7 days interval to get early emergence or as per prevailing environmental conditions. Then the subsequent irrigations should be applied at 8-9 days interval. Water should not rise from the edges of ridges. Half of the ridge should be covered by water. Excess or deficit in irrigation results in fast spread of tuber diseases and as a result the yield of potato is lower and shape of potato is also versed and the tuber may also scum to breaking. The last irrigation should be 15-20 days earlier than harvesting. Weed Control: Two types of weeds i.e broad leaved, itsit, krund,bathu,jangli palak and narrow leaved, deela and khabal grass. For control of weeds the weedicides like Metribuzin or Sencor or Zinker should be used after emergence of weed @200225 g/120 lit of water for 1 acre of potato yield. Rouging:All the undesirable or off type plants that are different from particular varity plants. Should be removed to keep purity for seed crop. The plants which show the symptoms of wilting or mosaic should be rouged out at 2-3 times during the season. First rouging should be carried out when plants are not close one and other and

after 38 days after sowing because some viruses i.e PVX spread from diseased to healthy plants. So in case of PVX, care should be taken out. Rouging should be carried out under shade condition i.e we should stand on those ridges where there is infection of virus diseases. The foliage showing mosaic type symptoms and wrinkled leaved, rolled leaves should be pulled out along with developing tubers and burry deeply in the soil. Rouging man should be careful during pulling of diseased plants that they may not be placed together with the healthy plants. Four ridges, two on each side can be rouged out.

Plant protection: Potato is a tender crop and a large number of pests and diseases infect the crop right from sowing up to harvesting. So watch full eye should be kept on potato crop condition during survey. First pest appears is the Jassid as name indicates it is swift green small pest having well developed wings that fly's and moves from one plant to the other. It sucks juice from the plant and the infected plants show swelling of veins. Incase of severe attack leaves assume the cup shape structure. Aphid also sucks the cell sap from leaves of potato plant. It is apparent from mid July up mid may. But the aphids are very important as they act as vectors spreading the viral diseases as aphid immediately transfers the virus into plants. Myzus persicae is a vector of PLRV. Aphis gossipy is a vector of PVY. Besides these pests, white fly may also suck sap from foliage and due to release of gelatinous mass fungus mold spreads and leaves become covered with black sooty mass.

Control: Pesticide from Dimethoate group or Cypermethrin group can be sprayed. Immidachloprid @200g/100lit. is most effective to

control aphid. Acetamiprid @150g/100lit. is highly effective for white fly.

Fungal Diseases of Potato

There are several fungal diseases of Potato but only important diseases will be touched rather in detail.

Late Blight of Potato

Disease appears in the late season of potato crop. This is one of the most important diseases in Pakistan. Its severe attack appears in cool and moist weather conditions. Late blight disease infects potato crop in the northern and few districts in Punjab like Sialkot, Lahore, Kaisor, Faisalabad and Sahiwal and if it is not timely controlled it can destroy the potato crop in few days. As the yield of potato is very much reduced. The disease travels to the young developing potato tubers which become the source of this disease. In case the infested tubers are stored in the cold store where is danger of rottening of tubers.

Causal Organism: Life Cycle:

Phtophthra infestans

This fungus attacks the potato crop planted in plain and hilly areas.

As this disease is primarily appears due to cultivation of diseased tubers (Blighted tubers). During harvesting process the diseased tubers are left over in the soil it

means that when we harvest the potato crop the young or infected potato tubers remain under the ground or these are not properly dug out. If these diseased tubers are not allowed to remain in the soil the chances of primary infection of disease are diminished. In other case if the diseased tubers are planted they also become the source of disease spread. It means that this disease is seed borne. When the potato crop is raised from infected seed the disease is transferred to the plant parts. Under favorable climatic conditions this diseased is transferred by wind. Temperature should be less than 18C and relative humidity more than 90%. These are the ideal conditions for the fast development of this disease. Under such conditions whitish powder appears on the underside of leaf. If during such climatic conditions is also rain and fog then this disease assumes epiphytotic conditions. It creates havoc. The particular symptoms are yellow hallow with powdery mass of fungus. When this disease approaches every part of the plant crop seems every part is scorched. This disease reduces the yield up to 40%.

Control:

trees.

Treat the infected seed with proper fungicide like Dithane M-45, Mancozib, Use of resistant varieties. Two varieties Cardinal and Dimant are not resistant but The potato should be cultivated in the field where there is no dense population of

Ultracol @ 29g mixed with 20g ash for a bag of seed potato 100Kg. some what tolerant as compared to Desiree.

Crop Rotation:
In the previous infected potato field potato crop should not be raised for 3 years. You should check the behavior of the disease in a particular field. It is better that

the potato crop should be planted after 3 years in the field. Same field should not be cultivated with potato again and again. So suitable crop rotation should be followed.

Pest Scouting:
We have to determine the EIL (Economic Injury Level). Pest scouting should be done in potato field to ascertain the Economic Injury Level. So that the proper fungicide can be used with correct dose at right time. The maximum earthing up should be done on the ridges so that the developing

tubers may not get in contact with the diseased pathogen. The ridges should be well covered with soil. The potato should be planted at proper time neither early nor too late. Irrigation should be applied according to the requirement of the crop. The crop

should not be over irrigated particularly when there is fog and cloudy weather there is chances of rainfall.

The excessive application of N and reduced application of P helps in spreading of The weeds should be eradicated by the use of weedicide. With the eradication of Even at the harvesting stage the disease spores are present on the leaves. It is better

the disease. So proper ratio of N, P and K should be used. weeds the alternate host plants should also be pulled out. that the haulm should be cut 10-12 days before the harvesting. So that the pathogen may not get access to the developing tuber. Spray of proper fungicide on the crop plants. Sencor/ Zinker/ Metribuzin @200-250g in 20 Litre water. Or Dithane M-45, Mancozib, Ultracol @200-250g /100L water for 1 acre. When the climatic conditions are favorable you should use the sprays of fungicide alternatively. Do not spray only a single fungicide again and again. When the Late blight symptoms appears on the potato crop then use systemic fungicide like Redomil MZ (Metalyxal+Mancozib) Or Redomil@ 225250g /100L water for 1 Acre

Dupoint or Curzate are some other fungicides available in market.

Early Blight of Potato

As the name indicates it appears in the early season of autumn crop as the temperature is higher. Similarly it also affects the potato crop especially in spring season when the temperature is favorable for this disease.

Causal Organism: Symptoms:

Alternaria solanii

This disease attacks the potato crop in plains as well as in hilly areas.

Brown spots with clear margins having concentric circles. Extent of loss is not more than 10-15%.

Favorable Climatic Conditions:

Warm and humid climatic conditions are favorable. As this disease does not spread so fast so there is no much loss. Early planted varieties in autumn have normal attack of this disease.

Control

Disease free seeds should be used. The crop should be harvested after the hardening of the skin. If the disease exceeds beyond the limit of 5% then you proceed for the spray of the

suitable fungicide like Dithane M-45, Sancozib, Mencozib, Ultrcol etc. If the disease is still progressing after the first spray then again repeat spraying of other fungicides

amongst the three one. These are the protective fungicides. The rate should be 250-300g per 100L water for 1 acre. The doses can be increased up to 400-500 grams. If the late blight disease is found with higher incidence than the early blight then the spray of fungicide against late blight will be suffice to control the early blight.

Black Scurf of Potato

It is a fungal disease caused by Rhizoctonia solanii. This is the soil borne pathogen. The infected seed give rise to growth of fungus with the emergence of plants from tubers. Its attack is initiated on the young growing plant. This disease is prevalent in hilly and plain areas. If the young plants are attacked there is development of the fungus near the level of stem. When the plant approaches to normal height there is development of black shank. The fungus makes lesions which are brown near the soil surface and these lesions turned black in form of scurf. Sometimes this fungus also infects the stolons. The roots of the plants are also affected means roots are reduced due which the plant remains weak. When the temperature assumes favorable tone for the development of disease the stem also gets rotted.

Favorable Climatic Conditions:

Low temperature and high moisture is favorable for the development of this disease.

Control:
Healthy seed of resistant varieties should be used.

If the soil is infected with the disease causing fungus Rhizoctonia solanii the Brassicol fungicide should be used at the rate of 10-15Kg depending upon the level of intensity of the disease. The seed should not be sown too much deep. Irrigation water should not be allowed to over flow the ridges. The harvesting of the tubers should be early rather than late. The harvesting should The potato should not be planted in the infested field at least for 3 years. Proper crop rotation should be followed.

not be delayed as the tubers lying underground are easily scum to the disease.

Fusarium Wilt
As the name indicates this disease is caused by Fusarium species. One of them is Fusarium oxysporum. It develops in the hot climatic conditions and inflicts serious damage to potato in both hilly and plain areas. This disease badly affects the potato crop. It is soil borne disease. At initial stage of its appearance the young growing plants get affected and they become weak and they start wilting and die. The particular symptoms of this disease are that in older plants the lower leaves turn their color to pale and these leaves becomes weak and hang. The stem of affected plants changed in coloration and the plant dies with in a few days. This disease progresses at 15-20C.

Preventive Measures:

Use of healthy seeds. Seeds should not be cut whole seed should be planted. Proper crop rotation should be followed. Do not plant potato crop in an infected field for at least 3 years. Only resistant varieties should be planted. Before cultivation the seed should be treated with Dithane M-45

Verticillum Wilt Causal Organism:

This disease is caused by verticilium sp.

V.alboatrum. and V.dahlia

Symptoms
The disease also spreads in hot weather conditions. It is uniformly found in hilly and plain areas. First the lower leaves are affected and there color is turned yellow. Characteristic symptom of this disease is that the few leaves of the plant become wilted and the rest remains green. Some times the symptoms are so clear that the leaflets of one side, after wilting, become dry and on the other side, remains green. If the diseased plant is cut vertically, the inner side of the stem appears brown. The passage of the sap is hindered and plant becomes dried.

Control:
Plant residues should be collected and burned after and before the

harvesting of the crop. Proper crop rotation should be followed. Badly affected varities, that are highly susceptible to disease should

not be used for sowing. In the infested field, the crop should not be planted. Resistant varities should be sown. Disease free seed should be used and seed should not be cut as some

farmers are forced to cut the seed due to the low availability of the seed.

Bacterial diseases of potato

Following are the bacterial diseases of potato

Bacterial wilt:
It is caused by pseudomonas solanacearum.It effect mostly spring potato crop.

Symptoms:
It appears on above and underground part of potato crop. This is found relatively in hot weather. In the above growing parts, first leaves and branches turn pale and dry, some parts show wilting. Potato particularly developing near point of root attachment become rotten. If affected tubers are pressed, gray material oozes. Some time creamy white decayed part of potato is visible. This attack is serious at 35-37C.

Preventive measures
1: proper crop rotation. 2: cut potato should not be sown. 3: sterilize the knife with 2 percent formalin soln. to cut potato. 2: common scab: It is caused by streptomyces scabies. It is found in Murree, Balakot, kaghan and Northern areas. Prevalent in plains of Punjab. Above parts do not show any symptoms. Developing tubers are affected. On tubers raised spots brown in colour develops. This shows scab spots; first they are of brown color then become dark brown. Spread fast at 25-30 C.

Control measures:

1: use healthy seed. 2: follow crop rotation. 3: Use Mancozeb 8 percent for affected seeds. 4: irrigate at proper time. 5: resistant varieties should be used.

3: Black leg:
It is also called soft rot. It is called Black Leg because it shows the symptoms of black leg develop on tuber. It is destructive because it affects the growing plant and potato tuber that develops on stolons. It is caused by Ervinia carotovora. Spp. If there is moisture in the soil. This pathogen develops at fast rate and can develop on stem of plant. As the result of attack the stem near the soil become black, that is why called Black Leg. If the rotted tubers planted in soil it completely rot and desirable plant population can not be achieved, because of loss, growing at the younger stage. First point of infection is attachment of tuber with soil. The leaves of diseased plant turn pale and turn brown. Under such cases plant die. Even if the seed tubers have some sort of injuries either in the field or cold storage, the attack of this disease can start. Besides the pathogen of this disease, they get transformed from the diseased to healthy plant through irrigation. Losses are higher under wet condition, rather hot or dry. The developing tubers escape from the disease. This disease spread quickly through infected tubers of especially injured ones. So control measures are concerned.

Control measures:

The resistant varieties of potato should be used. Cut Seed should not be used. If seed is cut then dipped in 2 percent formalin soln. to disinfect the knife. Incase knife is not sterilized; there are higher chances of transmission of bacteria into the seed tuber. Proper crop rotation for control of this disease should be used. The field should be lightly irrigated, when there are symptoms of this disease.

Major Potato Viruses

Potato leafroll virus (PLRV) causes an important disease of potatoes affecting the quantity and quality of production and may cause a crop to be ineligible for certification. Foliar symptoms of PLRV can be divided into primary and secondary infections. Primary infection results when an initially healthy plant is inoculated via aphids during the current season. Symptoms first appear where inoculation occurs. The upper leaves become pale, upright, and rolled and show some reddening of the tissue a round the leaf edges . The lower leaves may or may not have symptoms. Secondary infection occurs when an infected tuber is planted, giving rise to an infected plant. The lower leaves are severely rolled and leathery to the touch . The plant frequently has an overall stunted, upright, chlorotic appearance. The oldest leaves may show reddening on the margins or chlorosis. The upper leaves may not have obvious symptoms. Some varieties such as Russet Burbank are very susceptible to PLRV and to tuber symptoms of internal net necrosis . Many varieties grown in the Northeast are not subject to net necrosis. PLRV can be difficult to detect because foliar symptoms are not always obvious. Thus infected tubers or tubers with net necrosis may result from plants without visual symptoms. PLRV is transmitted in a persistent manner by several aphid species, the most important being the green peach aphid (Myzus persicae).In addition to infecting potato, the virus infects other solanaceous crops and weeds (tomato, tobacco, jimsonweed, etc.). Control consists of suppressing aphid populations with systemic and (or) foliar insecticides and planting certified seed. Potato virus Y (PVY) is one of the most important viruses infecting potatoes. It is readily spread by aphids in a nonpersistent manner as well as mechanically by human activity and may result in severely depressed yields. PVY is tuber borne

and can interact with other viruses such as PVX and PVA to result in heavier losses. Symptoms caused by PVY infection can vary depending upon the strain and potato variety grown. A rugose mosaic symptom is characteristic for some strains, but is most commonly ascribed to a mixture of PVY and PVX. Other strains produce a general mosaic or a hypersensitive (severe necrotic) reaction . Necrosis may progress to total leaf collapse, with the dead leaflet clinging to the stem. Some varieties with a strong hypersensitivity reaction display field resistance, and the progeny from such plants may be healthy. Besides infecting potato, PVY affects other solanaceous crops (tomato, pepper) and weeds (nightshade, groundcherry). Control depends on the use of disease-free seed, insecticides to reduce aphid populations, and mineral oil sprays to interfere with the aphid transmission process. Potato Virus X (PVX) is one of the most widely distributed viruses of potatoes because no symptoms develop in some varieties (latent mosaic), the full extent of damage with PVX is not recognized. Mixed infections of PVX with other viruses like PVY and PVA cause more damage than PVX alone. PVX is tuber borne and is readily mechanically transmitted by human activities. Tobacco, pepper, and tomato are additional hosts for this virus.

Minor Potato Viruses and Spindle Tuber Viroid

Potato Virus A (PVA), which causes mild mosaic, has a number of characteristics in common with PVY and belongs to the same virus group. Symptom severity will depend upon the strain, potato variety, and environmental factors. Many varieties (Katahdin, Kennebec, Sebago) reportedly react to infection with hypersensitivity (field resistance) as mentioned under

PVY. Control of this aphid-transmitted virus disease is through the use of disease-free seed, insecticides, and resistant varieties. Potato viruses S and M (PVS and PVM) occur in New York, but their importance in yield loss is uncertain. Both are aphid transmitted in a nonpersistent manner and are tuber borne. PVS is symptomless in most potato varieties, and many varieties express mature plant resistance. PVM may induce symptoms referred to as paracrinkle. These viruses may be most important when found as mixed infections with other viruses. Alfalta mosaic virus (AMY) occurs worldwide in potato, but is considered to be of little economic importance. Because AMV infections produce characteristic calico symptoms on the foliage, it is commonly referred to as "calico mosaic" . Aphids bring virus from reservoir hosts such as alfalfa and clover. Potato spindle tuber viroid (PSTV) can cause a destructive disease of potato and receives particular attention for certified seed production. It is often transmitted through breeders' progenies mechanically, as well as through pollen and true seed. PSTV consists of a small RNA molecule lacking the protein coat of viruses. Evidence suggests that chewing insects may be responsible for spread in nature, but humans and their activities serve as the principal disseminator. As the name implies, infected tubers may be spindle or oblong shaped or tend to be more rounded instead of the normal shape for a given variety . Prominent eyebrows are another important characteristic. Foliar symptoms on infected plants are not easily contrasted, making this one of the more difficult diseases to diagnose. The leaflets may be smaller and curve inwardly, giving a stiff upward growth habit . Sensitive tests (i.e., molecular hybridization) are now available to screen true seed and progenies to eliminate the viroid. Use of these tests and the

selection of certified seed are important steps in eliminating this disease from potato stocks.

Viral Diseases
In Pakistan there are various diseases of virus attack on potato. Among 7 viral diseases like PLRV, PVA, PVX, PVN, PVS and Moptop virus, every disease has its own importance. But among these PLRV and PVY are more destructive that not only effect yield of crop adversely but also seed quality is lowered. Some diseases spread through vector which play important role in spread of viral diseases. Attack of insect usually starts in the 1st week of January. Among the virus diseases potato virus has got significant position.

Major potato virus diseases

Sr.
1 2

Virus disease
PVX PVY

Mode of trans
Contact Aphis gossypii (Spring autumn)

Potential loss
10-25%

Importance
Moderate Major

40-70% 12-20% Major 40-70% 7-16% 40% 10-13% Moderate Minor

PLRV

Myzus persicae (Spring autumn)

4 5

PVA PVM

Aphid Aphid

PVS

Aphid

Spring 1020% Autumn 320%

Minor

Pot. Mop top virus (MPTV)

Fungus

Traces (Spongospora subperronaea)

Minor

PVY:
This disease spread through Aphid and through rubbing of the diseases plant with the healthy plant. It spread through insect and mechanical way. In Pakistan this disease is found in almost all the potato growing areas of Pakistan. If the population of Aphid is not controlled the spread of disease is progressive and fast. As the Aphid pick up the virus by stylet and with immediate feeding of healthy plant it transmit PVY. In early stage of disease the leaves of plant develop spotted lesions, rinckled on the leaves develop and after that streaks or specks appear on the stem. The main distinguishing symptom, the older leaves after wilting hang with the stem. In 3-4 weeks whole plants become wilted. In early stage if stem of the plant is broken then it give the sound of stem of the spinach. As result of attack of this disease. The disease plant produce small tuber and yield is considerably reduced. 3 types of this disease produce different symptoms including the patches of dark green and light green on the leaves. And the hanging of wilted plant and serious effected plant become stunted.

Control measures:
1: resistant varieties of potato should be selected. 2: small seed size with soft tissues should not be used. 3: rouging of disease leaf from shoot. If the plant is found with developing tuber, the whole plant should be uprooted and buried in barren soil. 4: control of insect pest which act as vector of disease. 5: seed should never be cut because transmission of diseases rapid with cutting. 6: spring crop should not be planted as it is very prone to attack of insect pest, particularly sucking pest. Mosaic Group of viruses:

PVX, PVS and PVM.

The mosaic disease of potato is found in early growing localities. There are many stains of viruses, some are not serious but few of them are destructive. If Mosaic attack is severe the yield loss is around 5-15%.

Symptoms:
Depend upon type of climate and the stains of viruses. The Mosaic symptoms, combination of dark and light green patches.

Virus X:
Creates 2 types of symptoms from mild to severe and other stains produce invisible.

Virus S:
It also produces Mosaic symptoms, but there are spots dark brown in color .

Virus M:
Midribs develop Necrosis.

Sucking pest of potato Jassid:

This is a sucking pest which attacks the spring as well as autumn potato crop It usually sucks the sap from the under side of leaf due to which the plants become week and veins of the leaves become swollen. incase the attack is severe, the potato crop shows the appearance of blighted plants and the yield is adversely affected. The nymph and adult of this insect inflict the damage.

Identification:
The color of the adult insect Is green. There are two dark spots on the forewings and on the top of the head.

Flourishing:
(period of activity) Flourish from April to December.

Damage:
It jumps from one plant to the other plant and develops green gelatinous material which gives the appearance of fungus mold on leaves.

CONTROL:
Important pesticides are Biphenthrin @ 300-500 mm. At mild stage apply @ 300 mm and in case of above economic level apply @ 300 mm500mm.Imidachloprid can also be used @ 200 ml- 250 ml/acre. in 100 lit. water. The under side of the leaves should be very well sprayed otherwise the

insects would remain flourishing. Dimethoate can also be used. Ethion and Thiodan can also be used but there availability can not be assured.

Aphid
It is also a sucking pest and sucks the cell sap from under sides of the leaves.

Chewing insect pests of potato

American boll worm:
This pest attacks both types of crops, autumn as well as spring. The larvae damage the foliage by feeding on leaves. In severe attack, the haulms seem to be devoid of leaves due to severe cutting. The color of the larvae is green and size of the caterpillar is up to 3.5 cm. It,s body has light brown or pale streaks on its lateral sides. Control: Chloropyrifos, Profenophos@1 lit/100 lit.water/acre.Cyhalothrin @250 ml/lit.

Army worm:
It attacks in gregarious fashion. The color of caterpillar is dark green. On the body of the insect larvae yellow strips are conspicuous. Male and female moths are also found in the field. The female lays eggs in small heaps underside the leaves which are covered with hair like structure. Among these eggs, the young caterpillar appears in groups and start chewing the leaves of the plants. In case of cotton, it also attacks the plant. The presence of brown or dark feces indicates the presences of this insect.

Control:

Profenophos @ 1 lit./100 lit. water/acre. Or Methomyl and thiodicar can be used @ 500 ml in 100 lit. water.

Potato tuber moth:

The caterpillar has green color and the body is of conical type. The female insect lays eggs on the eyes of tubers in store and the eggs hatch and the young caterpillar attacks the stored tubers. As a result the stored tubers start rottening. First after the emergence of the plants earthing up should be done.

Control:
Cypermethrin @ 250 ml/acre should be sprayed.

Cut worm:
As the name indicates this is the cater pillar that cuts the plant near soil level particularly at developing stage. The caterpillars remain hidden in day time and attacks young plants in night so this insect is called as cut worm. The body of the caterpillar has black strips and spots.

Control:
Carbaryl (seven dust) @ 10 % taking 5 kg and mixing in 15 kg well powdered soil. And this mixture should be tested at the evening If the attack is serious otherwise dont apply the pesticide. If it is possible, trace out the caterpillar near the cut plants by digging the soil and then these should be destroyed.

Mite:

In dry weather conditions this pest attacks the potato crop seriously. Mites are very small and globular and succulent. The under side of the leaves shoes small dots which are brown to yellow in color.

Control:
Three insecticides can be used for its control. Ethion @ 1 lit./acre. Triazophos or Hexythizox @ 500

Integrated pest management

IPM means collective steps taken systematically for the management of pest at proper time.

Principals of farmer lead IPM

Grow a healthy crop. Selection of high yielding varities having resistance against major diseases. Seed should be free from seed born diseases. Farmer should be aware of land preparation method. Farmer should adopt correct row to row and plant to plant distance. Farmer should know the balanced fertilizer dose. Farmer should have the knowledge about crop management, water management and weed management. For the control of pest, scientist should be consulted.

 All the predators, parasites and antagonist population should be kept in view as these are the defenders. Farmer should regularly visit the fields to review the pest and disease situation.

Diseases of General vegetables Musk melon Downy mildew

It appears in form of cottony moss spread on foliage twines tendrils and also on developing fruit. It develops under warm moist conditions .it can be controlled with spray of antracoal fungicide @ of 250g to 500g in 100 liter water. If it is not available diathane M-45 is used to spry at same rate. The latest arrival in market is of ellitte fungicide more effective at 250g per acre.

Powdery Mildew
In form of powder mass of spores foliage vines and fruits are covered with powdery mass. This is peculiar symptom of this disease. It usually spread under dry and warm weather condition. It is better controlled by sprying Baytan Foliar @50 ml.

Onion and Garlic

Purple blotch Mold Downy Mildew To control it Antracoal @ 500g and Ellitte @ 250g is used.

Chillies
Diseases are commonly found in field are Collar Rot ,Fruit Rot and Late blight. TO control fruit rot and Late Blight spray Ellite or Topcin instead of Ellite @250g.

Tomato Early Blight

This is the serious disease of tomato which also causes early blight of tomato. Causal Organism Alternaria Solani As name indicates it appears at early stage of tomato plant. The distinct characteristic of symptom are concentric circles. These are brown in color and later turned in dark brown and infects all parts of plant but concentric rings are irregular on leaves and stems. Control Only resistant varieties should be cultivated. Diathane M 45

 Antra Coal @ 500g If disease is progressing and attack is not serious then Antracoal can be applied at 2.5g to 3g per liter of water.

Late blight of Tomato

This disease also infect potato crop. It appears in late season of crop. Causal Organism Phytopthora Infestans Symptoms Development of water soak lesions covered with halo margin Under side of leaves presence of Mycelium Fungus Disease appears in cool and humid climatic conditions. Under severe attack lesions are found increasing inside can be effectively controlled with application of systemic fungicide Mencozab + Metalaxyl Redomil Gold 250g per 100 liter of water Egg Plant Diseases are blight. Protective fungicides of all kinds are used. Symptoms are same as given above.

Pumpkin

Root Rot disease.

Roots of plants get rotted and vascular system is clogged due to colonization. Causal Organism Rhizoctonia Solani Controlled when attack is on initial stage better fungicide is Elliete Lady Finger

Root Rot and Wilt disease.

These two diseases are soil borne Causal Organism Rhizoctonia Solani Fusarium spp. Can be controlled by following proper crop rotation so that the inoculums could not be built up. When there is problem of this disease same crop should not sown but tolerant varities are grown . Ellite @500g can be applied.

Cabbage
This vegetable is affected by two major diseases Wilt Leaf spot

Wilt
It appears at two stages early and other at middle stage.soil borne disease and sometimes appears in patches initially it can be control by pulling out diseased plants.so that disease can not spread to other plants through irrigation. Leaf spot Produces spots brownish in color which multiplies under favorable climatic conditions. This disease can be check easily by applying Diathane M-45 and topsin @ 2.5 g/acre.

Cauliflower
This vegetable has serious infection of Downy mildew a fungal disease. Cottony growth of fungus can observed under moist weather condition. Cottony spots are quite visible and by these spots disease can easily be recognized. Control Elliot 250g /100 liter of water /acre is used.

Carrot
Carotinoids are present abundantly in carrot and also rich in vitamin A. Its diseases are given below

Blight
Upper part of plant shows scortching symptoms. This seems as burnt by fire called as blight. On leaves more as compared to stem and branches.

Control

Mencozeb or antracoal @500g when attack is serious.

Root Rot
Also soil borne usually attacks on roots of plants. This disease is incited by soil borne fungi mainly Rhizoctonia Solani. It is better to control this disease through proper crop rotation. Other alternative is use of resistant varities.

Turnip
Downy Mildew
It is serious disease of turnip. Anyone previously mentioned fungicide can used.

Production Technology of MANGO

Delicious palatable fruit. It is king of fruits. Maximum mangoes can only be obtained by dew care of mango through management practices. To give good shape to mango tree is very important to give high yield.

AREA:
Mango is mostly cultivated in Multan, Muzafferabad, layyah, Sargodha, Bahawalpur. In Lahore division& Rawalpindi product ion is very low. in year 2002-2003 area mango is 99 thousand hectare &production was 10 thousand & 300 thousand tons. Future for mango cultivation in Punjab is bright . but mango gives more production as compared to other fruits most important variety , langra, Dusahri, Fajri summer bahisht , Anwar retool sensation, SS1, SS2, SS3, Chonsa, serooli, neelum.

IRRIGATION:
Weakly irrigation should given to mango tree during may June. But in winter done ones a month depending on weather condition. It is clear that during flowering initiation soil should keep rather dry. If excess irrigation is given during these stages new branches emerges instead of flowering emergence in this production is low in plain area of Punjab. In warm and dry weather condition interval should form. September & October should lesson because in these days flower bud on branches appear if water is apply in normal proportion maximum flower will emerge resulting higher fruit production.

FERTILIZER
Used for nursery FYM: 8 tones Urea: 175 kg SSP: 220 kg ISP: 85 kg

Immediate step to be taken is to prune the diseased dead branches & particularly those which are affected by malformation .Disease management relate two types of gardens. Rehabitation of old mango trees Management & nourishment of new mango plants Among all these steps pruning has got serious importance Pruning of small trees & cutting of branches should be done up to diseased portions. Water shoots are non productive and grows fastly should be cut off. Plant up to 3yrs should have the best shape If branch is enough long can be cut up to 12 inches Pruning time Before emergence when tree is not bearing fruit

 Early season of summer Timely application of fertilizer doses.

MACRO ELEMENTS
Nutrition doses of macro elements NPK & micro Zn, Cu, Fe, Mn, boron. TSP 2kg/plant Urea 3g/plant Potassium of sulphate 2kg/plant Gypsum 10kg/plant FYM 80kg

Micro nutrient
ZnSO4 240g/100 litre/8 plants (21% or 23%) CuSO4 200g Borax 240g FeSO4 100g

Pest management
Mango hopper

Adult&nymph both suck juice from tender leaf branches &inflorescence. Attacked plants show wilting. In case of severe attack of mango hopper fruit is not set properly.

Mealy bug
It is chewing type has developed mandibles. It cuts foliage but usually it sucks juice even nymph and adult from all parts of trees.

Major diseases of mango Black nose of mango: Brown spot of elliptical shape appears on leaves.
Spots clear on branches from top to lower side and withering starts.

Powdery mildew: Fungus disease has white to dark color of the powder.
Consist of spores of fungus &this powder spreads on tender & soft branches & also on fruits. Effective fruit setting mango anthracnose or wither tip. Drying of branches from upper to lower portion. This is clear fungal mass appear on branches & death of plant. Big trees of mangoes will suddenly if bark of stem of plant is deeply observed the burst bark is seen.From the affected portion black material oozes out.

Growth Calendar
In January (dormant period before flower initiation): mango growth & development February: initiation of flowering & emergence of new leaves March: flower initiation &development fruit setting beginning April: flowering &fruit initiation &setting

May: enlargement of fruit size June: enlargement of fruit continues early varieties in sindh ripening start July: in mid July harvesting of early varieties starts August: harvesting of late varieties September: same as in august October: vegetative growth starts new leaves emergence November: dormancy period starts December: again dormancy period starts

Plant Protection Measures

January: mango mealy bug February: hopper &scale & powdery mildew. March: spray fungicide powdery mildew, antracol, topsin, April: spray against anthracnose apply past on stems of plant it is multi purpose May: traps for fruit fly June: pest survey July: fruit fly population is studied September: hopper+ mites bark splitting & anthracnose may appear October: bordex paste application. November: dormant period December: before December bands against mealy bug applied near stem base of plant pesticide should dusted

Potassium sulphate: 82 According to soil survey in mango soil zinc is found deficient as 7.5% FYM should be used in month of December January 2 month before flower initiation First dose of chemical fertilizer applied in 3rd week of February 2nd dose is applied in April when fruit is set.

TIME OF FERTILIZER APPLICATION

FYM + phosphorus + potash Full dose in month of December & January 1/3 nitrogen in April when fruit size is of pear grain . 1/3 nitrogen after harvesting in august September After fertilizer application immediate irrigation is applied

ADVERSE EFFECT OF CLIMATIC CONDITION

1) Components 2) Theoretical level 3) Practical demonstration 4) Activities by trainee by hand 5) Survey report of vegetable crop 6) Collection of references Reference should be latest 2007-2008

Tick these new references which are applied in nature for controlling various diseases. Management of seed borne & soil borne disease of potato Commonly reported virus diseases if any new virus disease reported mention

Production Technology of Citrus

Importance of root stock if we develop root stock from rood there is chance of disease infection of fruit root caused by phytophthora then it was realized that root stock is propose solution so fruit production of citrus plants depends upon healthy suitable root stock on which it is gifted or budded choice of citrus root stock is of much importance owner of private nursery for their own interest purchased defective seed of khatti and sweet lime so following points are necessary to be consider while selecting root stock. i. ii. iii. iv. v. vi. Root stock should be such that grafted plants on such stock shod give good field and quality of fruit. It should compatible with soil and climatic condition Root stock should capable of giving commercial production for at least 30 days. Root stock should easily available At Root stock joint there should must bulging Root stock should resistant nematodes and virus diseases. Root stock can with stand heat and cold stresses.

vii.

Root stock could be vigorous and of higher pH vale because of size of alkaline it has been improved to investigation that if range common grape fruit and fritters early or if grafted over jatti khatti plant become taller and grow early.

These plants grafted over flying dragan and fortuneless plants because short in stature and they are used for high density plantation. Quality of fruit is much more dependent on root stock if navel orange grafted over kharna khatta fruits are rough and of inferior quality while true orange, Palestine sweet orange and jatti khatti drives full nutrients from soil and live better resistance against cold weather. Plants grafted on coleopteran mandarin and flying dragon and swing citrumelo have high degree resistance against and weather conditions. Important root shoot is jatti khatti

Characteristics of Jatti Khatti:

It grows at faster rate and becomes graftable in shorter period. Plants grafted over it become taller in shorter period. Its use is extensive and it fits well in soil with better drainage systems but plants over this root stock cannot withstand cold condition. Its root grows deeper, fruit size large production enough but quality is not so good. This root shoot is only suitable for common Malta and grapefruit.

Rung pur lime:

Plants grafted over this root shoot from fat and fruit of medicine size there is proper juice contents. Rangpur lime is more productive in those soils where jatti khatti dont give better performance particularly where soil pH is more this root stock is for malta, fruiter early grape fruit orange.

Kharna khatta:
It is Used in Peshawar as root stock. It does not suit well in plain areas because grafted plants are infected with diseases. This root stock is successful in heavy and moist soils. But in light soil ,its growth rate is slow and ensures good yield. In fruit soluble solids and acid contents are enough in proportion. These are important Fertilizer: Length: 1 acre size 220 feet width:190 feet

Fertilizer requirement Age pant

After 1 year 2 years 3 years 4 years 10 after

of Ammonium Single sulphate

476g 952g 1428g 1904g years 4760g

Surphate potash
100g 200g 300g 400g 1000g

Farm

yard

super phosphate
275g 550g 825g 1100g 2775g

management
10kg 20 kg 32 kg 40 kg 60 kg

To apply fertilizer leave first space from stem. Base and up to peripheral area of spread of tree should properly hold or inter cultured. Required doses applied and irrigated immediately. FYM to citrus plant must giving in sep.

Ammonium sulphate has ammonia + sulphate also and have lower pH that is very good for us and it has also 46% gypsum. ZnSO4 5g/L of H2O CuSO4 3g/L of H2O Iron sulphate Boric acid = 2.5g /L H2O

= 1g/L water

Manganese sulphate = 2g/L of H2O

Diseases
Foot root: fungal diseases basically but nematode tylenchuluss semipenetrans
causes injury in root. It permits entry of fungus in roots.

Citrus canker.
Topisn M-45 Diathane, Ellite @ 250g/L H2O.

Citrus anthracnose or withertip: Pest: White fly, citrus psylla, citrus lemon butter fly and citrus mites.. Causes of fruit fall
Stem End Rot
Fruit fly attack Improper crop cultivation and extensive irrigation because of intercropping. Prohibited are wheat maize sugarcane, cotton, berseem.

Proper crops are vegetables legume crops. Weak condition of plant Unfavorable weather condition Imbalance use of fertilizers sacks of jute should spread around tree which leave high degree of fruit fly damage sacks should wet with sugar solution and thereafter diptrex powder is spread. When fruitfly feed on sugar coated pesticides fruit they killed. Fruit trees should spread with diptrex @ 250g/100 H2O depending on intensity of fruitfly otherwise thinder @ 250g /L of water. Whenever there is attack of with trip the diseased branches should not including healthy parts. Other main reason of fruit fall in citrus is fluctuation of auxins then dichloro phenoxy acetic acid applied @ 15-20 mg/L.

Viral disease of citrus Citrus yellow vein clearing:.

lemon and sweet orange

Citrus tristiza:

lime, lemon, mandarin sweet orange sweet lime

Citrus ring spot: on grapefruit, rough lemon sour orange. Citrus greening:
sweet orange and mandarin.

Visit to Vegetable Research Area AARI, Faisalabad

We visited the research area of vegetable Research Institute AARI on 15-42009 under the supervision of Mr. Inam -ul Haq plant virologist, plant virology section.

Objectives:
To visually observe the lay out and growth of different summer vegetables. Identification and collection of diseased plant samples. To observe the insect pests of the vegetables.

During this survey we observed following crops:

Chilies
In chilies, we observed different research trials and observed varietal differences. we identified and observed following diseases in chilies:

Fruit rot:
Brown concentric rings and fruit rottening.

Powdery Mildew:
Powdery mildew is characterized by white powdery spots on the ventral surface of the foliage. Fruit size and number is also reduced.

Tomato
We visited tomato fields and tunnels and observed different diseases of tomato.

Fruit Rot: Wilt:

It is identified by dark brown concentric rings on the fruit.

In open fields, wilt was the most prevalent diseases. It is characterized by yellowing of foliage ,plant wilting and dying.

Cucurbits
In cucurbits we observed gourds and squashes. These summer vegetables were at early stage so there was no or very little fruiting in them. So no disease was so apparent to be identified.

Peas
Peas were at the last stage, so we only observed and identified powdery mildew in them. Powdery mildew is identified by pale brown withers and loose leaves.

Sweet pepper

In sweet pepper we observed Fruit Rot and Powdery mildew.

Carrot
We observed carrot crop at mature seeding stage.

Garlic
Garlic was also at flowering and seeding stage. Flowers are of white color and appear in form of round clusters. Along with these diseases we also observed and studied the Lay out system and insect pests of these vegetables. We also compared the varietal characters of different vegetables. This survey was very helpful in order to visualize the disease symptoms and observe the difference among different varities.

Tissue culture for virus free seed potato production

Plant tissue culture is the aseptic (free from microorganism) culture of any plant part in vitro. Tissue culture is utilized in the field of Biotechnology. Micropropagation is the rapid vegetative propagation of plants via tissue culture techniques. Micro-propagation permits the manipulation of physical and chemical conditions in the production of large numbers of high quality plant material potato within a short period of time. Following is the whole procedure for tissue culture.

MS media preparation

Total volume Agar Agar

1000mL 7.00grams

No. of tubes=100

Sugar Vitamin Iron. Macro nutrients Micro nutrients

30.00grams 10mL 10mL 100mL 1mL

Hoags Lands Nutrient

Macro Ca(NO3)2 H2O (Merck-A427020) MgSO4 7H2O (Merck-202 KNO3 (Sigma-P6030) NH4H2PO4 (Merck-A857424) Iron chelate (Sigma-E6760) or Na2 EDTA 0.045grams/Liter 0.94 grams/Liter 0.52 grams/Liter 0.66 grams/Liter 0.12 grams/Liter 0.06 grams/Liter

FeSO4 7H2O Micro H3BO3 (Riedel 11607) MnSO4 H2O (Riedel 14457)

28.00 grams/Liter 34.00 grams/Liter

CuSO4 H2O (BDH 5963960C) ZnSO4 5H2O (Merck A1141-81)

1.00 grams/Liter 2.2 grams/Liter 1.0 grams/Liter

(NH4)6 Mo7 O24 4H2O (Merck 4140118) H2SO4 (Come) 0.1mL mix with one Liter

5.0 mL/Liter

If pH is higher than required then we use acids (H2SO4 or HCl) to lower pH. If pH is lower than required then we use base (NaOH or KOH) to Increase pH.

Preparation of MS media Material/Apparatus:

Beakers of 250mL, 1000mL, 2000mL capacity. Measuring cylinder500mL, Conical flask (2000mL), 5mL pipette.Vitamins, Iron, Distilled water, Micro nutrients, Macro nutrients, Sugar, Gel gro (Agar Agar).

Method:
1. Weighing of Sugar. Sartoring balance was used for weighing. First I took a piece of paper made it clean from dust. I weighed it. It was 62mg. I weighed 60g of sugar for 2 liter solution. 2. I dissolved it in Distilled water. I used electric shaker until it dissolved completely. 3. I put Macro nutrients 200mL in this sugar solution. 4. Then I added iron 20mL in it.

5. I added Vitamins 20mL and Micro nutrients 2mL in it. 6. Now I made the volume of sugar solution up to 1 liter. 7. Now I took Gel gro 2g. I Put it in distilled water and dissolved it by boiling and stirring it. After the Gel gro was completely dissolved I made the volume up to 1 liter. 8. Now I mixed both of the solutions i.e. sugar solution of one liter and gel gro solution of one liter. In this way the total volume became 2000mL. 9. Now I checked the pH of the solution. Optimum pH requirement is (5.7). However the pH of my solution is (4.4). I put NaoH to shift it up to (5.7). 10.I used filler to fill the prepared MS media into the test tubes. The filler exactly filled 10mL of media in each test tube. I filled about 20 test tubes. 11. Packing of test tubes before sterilization a. I took a full size news paper and placed it below the test tube rack in which test tubes were placed. Put another quarter piece of paper below rack in the center, to make a rigid base. b. I put butter paper on the mouth of test tube. c. Then I put a cotton pad over it. d. Now I wrapped the news papers around the rack. e. I fixed the tape over its edges. 12.I used autoclave for the sterilization purpose. I first cleaned autoclave from the exterior as well as interior. I put fresh water about 5-6 liters in it because it uses steam for sterilization. Care should be taken that the heating rod is completely dipped. 13.Now I placed the packed test tube racks. 14.Polypropylene plastic sheets wrapped in a newspaper were also placed in it. Now closed the lid.

15.I fixed temperature 120C and Pressure 120lbs for 30 minutes. 16.After 30 minutes autoclave was automatically powered off. I waited for the complete release of pressure inside the autoclave. 17.When the pressure gauge showed zero pressure I opened the lid of autoclave and took out the test tubes and polypropylene plastic sheets.

Inoculation of Explant into MS media

Next step in potato tissue culture is to place the desired plant part on the prepared media for culturing. The media prepared and sterilized in the test tubes is to be utilized for inoculation.

INNOCULATION procedure
1. I sterilized my hands with spirit. 2. I sterilized the glass slab on which the whole operation was to be carried out. 3. I put out the plant from test tube. I placed it on the glass slab. I removed the foliage of the plant with the help of forceps and razor. 4. Now I used the forceps and blade to cut the plant into small pieces about 0.2mm in length. Each piece had at least one bud along with micro leaf on it. 5. I put the small piece of Explant into MS media. I took great care that the position of bud remained upward in direction. 6. I wrapped a sterilized polypropylene plastic sheet on the mouth of test tube with the help of rubber band. Precautionary measures During the whole operation keep the mouth of test tube inside the laminar flow cabinet, towards the flow of air.

 During inoculation process care should be taken to keep bud position upward. Use moth mask to cover your mouth and nose during whole operation so there is less chance of contamination.

After understanding the procedure I inoculated 10 plants of SH-5 Varity.

Monitoring of plants in the Growth room:

I placed inoculated test tubes in the test tube stand and placed them in growth room. I labeled the test tubes with varity name and date of inoculation. The temperature in the growth room was maintained at 21-25 c and humidity was 80%.I monitored the growth of the plants and collected data at 10 days interval about root length, shoot length and no. of nodes. Following is the data of SH-5 varity. Sr.no:
No. of 1 2 3 4 5 6 7 8 9 10 Avg. nodes 3 2 3 2 2 2 4 2 3 3 2.6

23-4-2009
Root Length 0.7 (cm) 0.3 0.5 1.3 0.5 0.75 1.0 0.5 0.3 0.5 0.63 Shoot length 1.7 (cm) 1 1.5 1 0.5 1 1.3 1.2 1.1 1.4 1.17 No. of nodes 7 4 7 5 6 7 8 6 7 7 6.4

3-4-2009
Root length 1.3 (cm) 1.2 1.5 3.3 1.5 2.5 2.7 1.7 3.9 1.8 2.14 Shoot length 3.7(cm) 2.5 3.5 3.7 2.5 3.5 5 4.7 5 5.7 3.98 No. of nodes 10 7 12 10 7 10 11 12 10 8 9.7

13-4-2009
Root length 2.5(cm) 2.0 2.7 4.5 2.8 3.2 3.5 3.0 4.7 3.3 3.22 Soot length 4.5(cm) 3.2 4.7 4.2 4.0 4.2 7.0 5.2 6.5 6.9 5.54

Transfer of plantlets from test tube to sand trays

After the full growth of plants in the test tube that is when plants touch the rim of the test tube, these plants are transferred to the sand trays. The purpose of this activity is to acclimatize or harden the plants. The test tube plants of SH-5 varity were ready to transplant in the sand trays in 30 days. The procedure of transfer is as under. 1. I removed polypropylene plastic sheet from test tube. 2. I gave irrigation to the sand so that it became moist for the ease of making hole in it. 3. I observed the length of plantlet root and made a hole of similar size in the sand with the help of forceps. 4. I used fungicide Dithane M45 2% solution as a disinfectant. 5. I took out the plant from test tube with the help of forceps. I gripped it from middle portion neither from roots nor from apical portion, otherwise damage might occur to the plantlet. 6. I dipped the plantlet in water to remove the MS media contents. I gave at least 3 washings with water. 7. I dipped the plantlet in fungicide solution to disinfect it. I gave 3 washings of fungicidal solution. 8. Now I gripped the plant from its roots. I Put its roots in to already made hole. 9. I hardened the sand near the roots of the plantlet with the help of forceps or hand. 10.I irrigated the sand for proper establishment of the plantlet in the sand. 11.I labeled the pot. On label I mentioned the Variety name and the Date of transfer. The plantlets would be kept in sand trays for about 7-10 days depending upon the variety. After that duration they would be shifted in to the pots filled with soil instead of sand.

Transfer of plantlets from sand trays to pots

After 10 days plants were shifted to the pots.First I pulled out plants from the sand tray with the help of forescep and dipped in Dithane M45 soln. I made a hole in the pot with the help of forescep and inserted the plant in that hole and filled the hole with soil. The media in the pots was composed of common soil, farm yard manure and peat moss. After transplantation, I irrigated the pot and labeled with date of transplantation and varity name. In about 2-3 months, depending upon the varity micro tubers are produced in the pots which are further planted in the tunnels to produce minitubers.

Mini tuber production in the tunnels

Micro tubers are planted in the tunnels.Proper conditions of temperature and humidity are maintained in the tunnel. These micro tubers produce mini tubers which are used for seed purpose.

Potato tissue culture to produce virus free plants

ENZYME LINKED IMMUNO SORBANT ASSAY

VIRUSES
Virus is submicroscopic, infectious, intracellular entity, containing RNA or DNA genome and protein coat.It may or may not have lipid envelope, may have broad or narrow host range and its replication involves eclipse (breaking a part of virus particles) and reassembly.

Composition of viruses
The two major components of a virus are nucleic acid and protein. All viruses have their genetic information in the form of nucleic proteins are the major structural component of viruses. Coat protein (capsid) is the protective shell for the nucleic acid in

acid which is either RNA or DNA.

virion.

Detection and identification methods for plant viruses

Due to obligate nature of plant viruses, detection and identification methods used are quite different from those applied to detect fungi or bacteria. There are some simple methods of virus detection.

 Biological Serological Physio-chemical.

Serological tests
Tube precipitation test Ouchterlony double diffusion test Latex agglutination test ELISA-Enzyme-linked-immunosorbent-assay

Serological Methods
When an animal is injected with a pathogen, whether it be a virus or bacterium or a foreign protein, specific proteins are produced in the blood serum of the animal. these proteins are known as antibodies Combine specifically with the substance injected, known as antigen. Blood serum containing antibodies is called antiserum. Antibodies bind with antigen to produce a precipitate and is the basis of serological tests for viruses.

ELISA
The sensitivity of detection of antigen-antibody reactions can be increased by attaching to either of the two reactants a label that can be detected in minute quantities such as enzyme. The use of enzymes for labeling antibodies was used for localizing antigens. When the reactants were attached to a solid phase, enzyme immuno -assay was particularly suitable for the quantitative measurments of antigens and antibodies. The presence of bound enzyme is revealed by a chromogenic substrate, which is initially colorless but yields a colored product after enzymatic degradation. Besides a visual scoring for field screening of virus infected sample, accurate quantitative readings can be made with a spectrophotometer.

TYPES OF ELISA
Direct or double antibody sandwich (DAS) ELISA Indirect or direct antigen coated (DAC) ELISA Triple antibody sandwich (TAS) ELISA

DAS ELISA
Specific immunoglobin (IgG) extracted from antiserum are used for coating the plates Enzyme conjugate has to prepare with some IgG for the detection of antigen.

Antigen is sandwiched between IgG and IgG conjugate enzyme the method is generally known as double antibody sandwich method.

ADVANTAGE
This ELISA is relatively quick and avoids potential problem of cross reactivity of the secondary antibody with components in the antigen samples. Mainly used for distinguish of virus and to develop serological relationship of viruses.

DISADVANTAGE
It requires specific conjugate antibody for each virus to detected.

PREPARATION OF ELISA BUFFERS ANTIBODY COATING BUFFER

pH=9.6 Store at 4OC

CHEMICALS

Sodium carbonate =0.1598g/100mL Sodium bicarbonate=0.290g/100mL Sodium azide (NAN3) =0.020G/100mL

WASHING BUFFER
pH=7.4

CHEMICALS
Sodium chloride=40g/500mL Sodium bibasic phosphate=5.75g/500mL Potassium monobasic phosphate=1g/500mL Potassium chloride=1g/500mL Tween20=2.50mL/500mL

VIRUS EXTRACTION BUFFER

pH=7.4 Store at 4 c

CHEMICALS

NACL=8G/100mL KH2PO4=2G/1000mL NA2HPO4=1.15G/1000mL KCL=0.2G/1000mL NaN3=0.2G/1000mL PVP=20g/1000mL TWEEN 20=0.5mL

CONJUGATE BUFFER
pH=7.4 store at 4oc

CHEMICALS
NACL=8g/1000mL KH2PO4=0.2G/1000ML NA2HPO4=1.15g/1000mL KCL=0.2g/1000mL TWEEN=0.5mL/1000mL PVP=20g/1000mL EGG ALBUMIN (20%) =20g/1000mL NAN3=0.2g/1000Ml

SUBSTRATE BUFFER
pH=9.8 Store at 4 c

CHEMICALS

DIETHANOL AMINE=97 mL/1000mL NAN3=0.20g/1000mL PNP=1 TABLET (20g)

PROCEDURE;

STEP 1

Coat ELISA plate with diluting of antibody at ratio of 20L/20mL Mix them and load the ELISA plate @ 200L in each well. Incubate at 4oc for overnight.

in antibody coating buffer.

STEP II

Next day wash the ELISA plate with washing buffer 3 times. STEP III

Collect sample to be tested (infected leaves with sugarcane mosaic Grind with virus extraction buffer at ratio of 1:1, 1:10, 1:20 and so Load sample after washing @ 200L/well along with positive and Cover the plate and incubate in refrigerator at 4oc for overnight. STEP IV

virus.

on depending on sample.

negative control

Take out the ELISA plate and observe it Wash the ELISA plate with washing buffer. Drain out all samples from plate. This material contain cells, Prepare the conjugate anti sugarcane mosaic virus antibody.

proteins, plasma, all other debris and solution.

mix well the secondary conjugate IgG and conjugate buffer @of 20L in 20mL conjugate buffer.

With the help of multichannel pipette, we do coating of Load the ELISA plate with secondary IgG @ of 200L/well.

conjugate antibody.

STEP V

Incubate at 4oc for overnight. Pour of the enzyme conjugate and give washing with Load Elisa plate with substrate buffer 200L/well. Place the plate at room temp for to 1 hour. reaction start. Use sodium hydroxide 3M sol to stop reaction @of

washing buffer.

50L/well.

P-niyrophenyl changes to P -nitrophenol after reaction.

RESULTS

Yellow color will start developing in positive control first. No color development in negative control. Record data on the basis of color We can also use ELISA reader for reading. 1 tablet of PNP(20mg) in 20mL water for 1 ELISA plate.

SUMMARY
My selected discipline for internship was TISSUE CULTURE. So I was attached to TISSUE CULTURE LABORATORY in Directorate of Horticulture Sciences AARI, Faisalabad. But after few days, I was transferred to potato tissue culture laboratory under the Plant Virology section due to no availability of staff and equipment in Horticulture Institute. So I started my internship under the supervision of Mr. INAM UL HAQ (Plant virologist) in Plant virology section.Mr. inam ul Haq delivered us lectures on importance and uses of potato, seed potato production technology, Diseases of potato including Late blight, Early blight, Black scurf, Fusarium wilt, Verticilium wilt, Bacterial wilt, Common scab, Black leg, PVY, mosaic group of viruses, PLRV and Mop top. Insect pest of potato including Jassid, Aphid, Mite, American Boll Worm, Army Worm, Cut Worm and Potato Tuber Moth, IPM for potato, Diseases of general fruits and vegetables ,fruits included Mango and Citrus while Vegetables included Muskmelon, Onion, Garlic, Chilies, Tomato, Egg plant, Pumpkin, Lady finger, Cabbage, Cauliflower, Carrot and Turnip. We also visited Vegetable Research Area to visually observe different experimental trials and to study varietal characters of different vegetables. We observed Tomato, Cucumber, Chilies, cucurbits; peas and garlic .We also studied production technologies of mango and citrus. Practically we done tissue culture for Virus free seed potato production for six varities that are CARDINAL, DESIREE, DIAMONT, SH5,FD8-1, FD1-10.We prepared MS media, pored into test tubes and inoculated and placed in growth room at controlled temperature, light and humidity. We monitored them weakly and collected data for no. of nodes, root length and shoot length in the test tube. After 15-30 days, depending on the varity, the plants were ready to transplant in the sand trays in glass house. After

transplanting in the sand trays, we monitored their growth in the sand trays and transplanted them in the pots in glass house. In the pots, these plants produced micro tubers that is further used to plant in the tunnels to get mini tubers.We collected and compared data of all these 6 potato varities. Second major activity was ELISA.WE performed ELISA test for Water melon Mosaic Virus. First its procedure was demonstrated by Mr. Inam ul Haq (supervisor) and then we performed this test under the supervision of Miss Asia Batool (ARO). We also studied and collected relevant literature from library (AARI) and internet as well. And this was a very useful activity in order to increase our knowledge, broaden our vision and to know the international trends. This literature was also discussed in the class with our internship supervisor and other colleagues.

REFERANCES
Agrios, G. N. 2005. Plant Pathology, Fifth Edition Academic Press, N.Y. London Ahmad , S. I. and A. R. Bhutta. 1989. Major diseases of potato and their control. Prog. Farm. 9(3):20-25. Anonymous. 2007, Pakistan Agriculture Research Council, 2006-2007. Govt. of Pakistan Barnes, E.H. 1979, Atlas and Manual of Plant Pathology, Plenium press N.Y. Blackman R. L, Eastop, V. F. (1994). Aphids on the World's Trees: An Identification and Information Guide. CAB International: Wallingford. Chaudhary , J.A., S. M. A. Shah and A. Hissain. 1992. Performance of seed potatoes produced in the high and mid hills of Kaghan in Pakistan. Sarhad j. Agric. 8(1):29-32. Chaudhary, J. A., S. M. Mughal and N. A. Khan. 1990. Impact of improved seed sources on growth characteristics and yield of potato . Sarhad j. Agric. 6(3):249-253. Clark, M. F. and A. N. Adam. 1977 . Characteristics of micro plate method of enzyme linked immunosorbant assay for the detection of plant viruses. J . Gen. Virol. 34:475-483 Gray,S., K. Perry and P . Baldauf.2003. The genetic diversity of PVY in the main potato cropland pr4evalence tuber necrosis. Potato Board Report. P . 1-5.

Jafarpour, B., D. S.Teakkle and J . E. Thomas. 1986. Incidence of potato virus S and X and potato leaf roll virus in potatoes in Queensland. Australian Plant Path. 17(1):4-6. Kamal, M. and S. M. Moghal.1968. Studies on plant disease of South West Pakistan. ARI, Tandojam. Agriculture University, Tandojam Khan JA, Aminuddin, Raj SK, Singh BP (1998). Detection of plant viruses-biotechnological and molecular advances. Indian J. Exp. Biol. 36:546552. Mughal, S . M. and S . Khalid. 1985. Virus diseases in relation to potato production in Pakistan. In: Potato in Pakistan. P .154165.PARC,Islamabad. Nantulya VM (1991). Molecular diagnosis of parasites. Experientia 47:142-145. Picozzi K, Tilley A, Fvre EM, Coleman PG, Magona JW, Odiit M, Eisler MC, Welburn SC (2002). The diagnosis of trypanosome infections: applications of novel technology for reducing disease risk. Afr. J. Biotechnol. 1: 39-45. Rocha-Pena MA, Lee RF (1991). Serological techniques for detection of citrus tristeza virus. J. Virol. Methods 34: 311-331. Schneller, A.R. P.,K. L. Perry and S.M. Gray . 2003. Potato virus survey in New York and Maine and characterization of PVY isolates. P.2004-0002. NEA Report. P.1 Solangi, G. R.,S.M. Mughal and S. D.Khanzada. 1983. Identification of some viruses infecting Solanaceous hosts in Sind. Pak. Bot. 15:19-24. Tuite, J, 969, Plant Pathological Methods; Fungi and Bacteria, Burgess Publishing Company, Minneapolis, Minn.

Van Regenmortel MH (1984). Monoclonal antibodies in plant virology. Microbiol. Sci. 1: 73-78.