Citrus Pathology PLP 5115C Fall 2017

Meeting times: Mondays at 1pm to 4pmInstructor: Dr. Nabil KillinyOffice: Building 7103, Room 5, Citrus Research and Education

CenterOffice hours: Drop-in office hours Mondays 9:00-10:30, or by

appointment, or email anytime.

Course Schedule - Fall 2017Due Date* # Topic Instructor August, 21 1 Course overview

• Overview of citrus diseases• History of major citrus diseases.

Killiny

August, 28 2 Epidemiology of citrus diseases DewdneySeptember, 4 Holiday September, 11 3 Fungal Diseases of fruit and foliage Dewdney September, 18 4 Management of fungal and bacterial diseases Dewdney September, 25 5 Characteristics of viral and viroid pathogens LeveyOctober, 2 6 Citrus viral and viroid diseases

Case study1: Citrus leprosis (Citrus leprosis virus)Case study2: Viroid diseases

Brlansky

October, 9 7 Citrus viral diseases Case study: Citrus tristeza (Citrus tristeza closterovirus)

Dawson

October, 16 8 Citrus Root PathologyCase study 1: Citrus nematode (Tylenchulus semipenetrans)

Duncan

Due Date* # Topic Instructor October, 23 9 Citrus Root Pathology

Case study 2: Phytophthora Diseases. (Foot rot, root rot, brown rot, gummosis, and Phytophthera-Diaprepes (PD) complex)Case study 3: Huanglongbing (Candidatus Lebribacter asiaticus)

Evan

October, 30 10 MID-TERM EXAMNovember, 8 11 Characteristics of bacterial pathogens

• Eubacteria• Mollicutes

Killiny

November, 13 12 Citrus Bacterial Diseases Case study: Citrus stubborn (Spiriplasma citri)Case study: Citrus variegated chloroses (Xylella fastidiosa)

Killiny

November, 20 13 Citrus Bacterial DiseasesCase study1; Citrus greening (Candidatus Liberibacter asiaticus)Case study 2: Canker (Xanthomonas citri)

Wang

Due Date* # Topic Instructor November 27 14 Citrus responses to various pathogens

• Phytohormones• Metabolic responses• Volatile organic compounds• Micro-RNA• Pathogenesis-related proteins

Killiny

December 4 15 Genetic manipulation for disease resistance• Transgenic plants• Virus-induced gene silencing (CTV)• Expression vectors (CTV)• CRISPR/Cas system• Citrus breeding

Killiny

December 10 Final Exam

Reading Materials Nothing assigned for now! Some handouts from lecturers Compendium of Citrus Diseases Plant Pathology - Agrios Essential Plant Pathology- Schumann and D’Arcy Plant Virology – Hull Plant Bacteriology – Kado

Concepts of Plant Pathology What is plant pathology? Pathology: the study of the nature of disease or any

abnormal condition in an organism. Plant Pathology is “1. the study of living organisms and the

environmental conditions that cause disease in plants 2. the mechanisms by which these factors produce disease in

plants , 3. the interactions between disease-causing agentsand the diseased plant, and 4. the methods of preventing orcontrolling disease and alleviating the damage it causes”

What do Plant Pathologist Study? Diseases caused by fungi, bacteria, mycoplasmas, parasitic

higher plants, viruses, viroids, nematodes and protozoa Also plant disorders caused by excess, imbalance, or lack of

physical or chemical factors (e.g. moisture, temperature and nutrients)

But not plant damage caused by insects, humans or animals

Plant Pathology uses basic knowledgeof: Botany, mycology, bacteriology, virology, nematology, plant

anatomy, plant physiology, genetics, molecular biology,genetic engineering, biochemistry, horticulture, tissueculture, soil science, forestry, chemistry, physics,meteorology, and other branches of science.

Uses advances of other sciences to solve plant pathologicalproblems.

Plant pathology attempts to increase our knowledge of thecauses and development of plant diseases

Practical goal is: to save produce that is destroyed by plantdiseases to make it available to feed and cloth the world.Done by control and management of diseases.

Concept of Disease in Plants What is a healthy plant? When it can carry out its physiological functions to the best

of its genetic potential Functions include what? normal cell division, differentiation, development;

absorption of water and minerals from the soil and translocation throughout the plant; photosynthesis and translocation of the products to areas of utilization or storage; metabolism of synthesized compounds; reproduction; and storage of food supplies for overwintering or reproduction.

Plant disease (one definition): whenever plants are disturbedby pathogens or by certain environmental conditions andone or more functions are interfered with beyond a certaindeviation from the normal

The primary cause of disease are either pathogenic livingorganisms (pathogens) or factors in the environment.

Definition of symptoms: Evidence of the reaction of theplant to the pathogen or materials that the pathogenproduces (or environmental factors). Macroscopically ormicroscopically.

Types of Function Interference Root rots: root infection Vascular wilts and cankers: interference with the vascular

system Leaf spots, blights and mosaics: infection of foliage Viral, phytoplasma and systemic bacterial infections:

interferes with translocation of photosynthetic products Bacterial and fungal blights: interferes with flowers Fruit rots: causes reduce reproduction or storage of reserve

foods for the new plant

Hyperplasia or hypertrophy: more cells produced or largercells produced resulting in nonfunctioning or abnormal cellsor abnormal cell growth; often diverting food materials fromthe rest of the plant

In all cases of disease the physiology of the plant is affected Now let’s define disease: The malfunctioning of host cells and tissues that results from

their continuous irritation by a pathogenic agent orenvironmental factor and leads to the development ofsymptoms

Is disease a condition? Yes, a condition involving abnormal changes in the form,

physiology, integrity, or behavior of the plant that may resultin the impairment or death of the plant or its parts.

How do pathogens cause disease in plants? 1. weaken the plant by absorbing food from the plant, 2.

killing or disturbing the metabolism of the cells via toxins,enzymes or growth regulating substances, 3. blocking thetransportation of food, minerals, and water, 4. consumingthe contents of the host cells

Classification of Plant Diseases I. Infectious or biotic plant diseases (type of pathogen)

caused by 1. fungi, 2. prokaryotes (bacteria orphytoplasmas), 3. parasitic higher plants, 4. viruses andviroids, 5. nematodes

II. Disease symptom: root rot, canker, mosaic, foliage, leafspot, wilt, fruit disease, post harvest, etc.

III. Field crop disease, fruit tree disease, forest disease, turfdisease, ornamental disease, etc.

Question to class:Which is the most useful?

History of Plant Diseases

Begins with fungal diseases OldTestament lists blasting and mildews Greek philosopher Theophratus (370- 286 B.C.) first to

study and write about diseases of trees, cereals and legumes. Romans: created a special rust god, Rubigo, to protect them

from cereal rusts. Each spring celebrated Robigalia 1729 Micheli: His Nova plantarum genera (1729) was a major

step in the knowledge of fungi. 1st to point out that fungihave reproductive bodies (spores) (dust particles taken froma fungus reproduced the same kind of fungus

1755 Tillet: black dust from bunted wheat to seed fromhealthy wheat and produced bunt in the plants.

1807 Prevost: bunt disease caused by a fungus Debary 1853: smut and rust fungi are the causes not the

results of plant diseases. Remember he was a microscopist Brefield 1875, 1883, 1912: introduced techniques for

growing microorganisms in pure cultures. Assisted by theaccomplishments of Koch, Petri and others

1878 Downy mildew of grape was introduced into Europeform the U.S. Spread rapidly. Millardet noticed that vinessprayed with copper sulfate and lime to deter pilferersretained their leaves during the season. “Bordeaux mixture”

1882 Ward: coffee rust in Ceylon warned againstmonoculture of crops

Historical Perspectives withDifferent Causal Agents

Bacteria in Plant Disease 1876 Pasteur and Koch proved anthrax was incited by a

bacterium (Anthrax is an infectious disease of both humansand other animals caused by bacteria called Bacillus anthracis.Infection in humans most often involves the skin, thegastrointestinal tract, or the lungs).

E. F. Smith 1895: bacterial diseases of plants; bacterial wilts ofcucurbits, ornamentals, crucifers. Established bacteria asphytopathogens

1977 Chilton: Crown gall bacterium transforms normal plantcells into tumor cells (anyone know the causal agent?)

1972 Windsor and Black: Rickettsialike organisms in thephloem of clover with clover club leaf.

1973 and after: Fastidious bacteria in the xylem of Pierce’sdisease of grapes, phony peach, and others (who cultured it?)

Nematodes in Plant Disease 1743 Needham within wheat galls 1913-1932: Cobb series of studies on plant parasitic

nematodes

Viruses in Plant Diseases 1886 Mayer: tobacco mosaic: juice from plants was

infectious. “contagium fluidium” 1929: Holmes: local lesions could be used to measure the

number of virus present in plant sap prep. 1935 Stanley: precipated the crystalline protein from

infected tobacco plants and showed that the virus was anautocatalytic protein that could multiply in living cells(Nobel Prize)

Bawden in 1936: virus was protein and RNA 1939 virus particles first seen with the electron microscope 1956 Gierer and Schramm: protein could be removed from

the virus and that the nucleic acid carried the genetic info

1962 Agar double diffusion serological test for viruses 1977 ELISA test for viruses 1975 monoclonal antibodies Nucleic acid studies: 1936 to 1977 1971 Diener determined that potato spindle tuber disease

was caused by a viroid (What is a viroid? Is it unique toplants?)

1980 Cauliflower mosaic virus, a circular double strandedDNA, was first sequenced (8,000 base pairs)

1982 complete sequence of single strandedTMV was done

Protozoa and Phytoplasmas 1909 Lafont observed flagellate protozoa in latex bearing

cells of laticiferous plants of Euphorbiaceae 1931: Stahel found flagellates infecting phloem of coffee 1963 Vermeulen showed evidence of flagellates in coffee and

in 1976 they were associated with coconut and oil palms 1967 Doi in Japan observed mycoplasmalike bodies in the

phloem of plants infected with leafhopper diseases 1967 Ishiie and showed that symptoms disappeared

temporarily if treated with tetracycline antibiotics 1971 Davis et al observed spiroplasmas in corn stunt disease

Diagnosis of Plant Diseases Pathogen or Environment: Look for sign of the pathogen or

symptom ( already described) which might infer pathogen Infectious diseases: diseases caused by pathogens Characterized by their presence on or inside the plants Parasitic higher plants: dodder, mistletoe, witchweed or

broomrape growing on the surface Nematodes: presence on or in a plant or in its rhizosphere,

but must be distinguished from nonparasitic ones;taxonomic

Fungi: must determine if it is a parasite or saprophyte;morphology of its structures; taxonomy and reports ofpathogenicity on plant species

Bacteria: diagnosis of the disease and the ID of the bacteriumin based primarily on symptoms, constant presence of thebacterium and absence of other pathogens; culturing,selective media and taxonomic characterization; serology,PCR, fatty acid analysis; avoidance of looking at secondaryorganisms; fastidious bacteria difficult (small and sometimesin low numbers) and are difficult to culture.

Phytoplasmas and spiroplasmas: usually cause stunting andyellowing of plants and other specific symptoms; electronmicroscopy and PCR is used; P’s not culturable, S’s are;often graft transmissible and sensitive to antibiotics, insectvectored

Viruses and viroids: distinctive symptoms on hosts and oftenidentified this way; virus transmission tests to specific hostsby sap transmission or grafting; transmission by certaininsects; serological test available for many, PCR, electronmicroscopy, light microscopy of viral inclusions;electrophoretic and PCR tests for viroids;

Noninfectious diseases: lack or over abundance ofsubstances, environmental factors (look for specificsymptoms)

Koch’s Rules (Postulates) Pathogen found associated with the disease in all diseased

plants Pathogen must be isolated and grown in pure culture on

nutrient media and its characteristics described or on asusceptible host plant (obligate parasites) and it appearanceand effects recorded

Pathogen from pure culture must be inoculated to healthyplants and must produce the same disease symptoms

The pathogen must be reisolated from the disease plant andit characteristics must be the same as in step 2.

What about viruses and viroids? What about a positive PCR and accompanying sequence

determination? Is this enough? What about circumstantial evidence?

Break

Parasitism and Disease Development Infectious diseases: result from infection of a plant by a

pathogen Parasite: an organism that lives on or in some other

organism and obtains its food from the later Parasitism: relationship between a parasite and its host Is a plant parasite a pathogen? Terms to consider: symbiosis and pathogenicity

Symbiosis and Pathogenicity Symbiosis: mutual benefit to parasite and host Pathogenicity: the ability of the parasite to interfere with one

or more essential functions of the plant with parasitismplaying an important but not always the most importantrole.

Considerations Obligate parasites Nonobligate parasites Facultative saprophytes Facultative parasites In N. America: 8000 species of fungi cause 80,000 diseases,

200 species of bacteria and 75 phytoplasmas, more than 500viruses attack crops.

Development of Disease What is necessary for a plant disease to occur? Plant Pathogen Susceptible plant Environment for disease to develop or express itself Called the “DiseaseTriangle” Variability of each component can change the degree of

disease severity Could there be an additional component of the triangle?

Stages in Development of Disease:

The Disease Cycle Infectious diseases have more or less distinct events in the

succession that lead to the development and perpetuation ofthe disease and the pathogen

This chain of events is the disease cycle May correspond to the pathogen life cycle but not always Disease cycle involves changes in the plant and the

symptoms of the plant as well as in the pathogen and spanstime within the growing season and from one season to thenext

The Disease Cycle Inoculation Penetration Establishment of infection Colonization and growth Reproduction of the pathogen (could be further growth) Dissemination of the pathogen Survival of the pathogen in absence of the host(overwintering or oversummering)

Inoculation Pathogen contact with the host- inoculum Spores, sclerotia, fragments of mycelium, individuals ofbacteria, phytoplasmas, viruses, viroids, etc. Propagules: single units of inoculum of a pathogen Types of inoculum: primary, secondary Sources of inoculum: debris, soil, tubers, transplants, seed Landing or arrival of inoculum: wind, water, insects

Prepenetration Phenomena Germination of spores or seeds Hatching of nematode eggs Attachment of pathogen to the host plant Recognition between host and pathogen

Penetration Direct penetration through intact plant surfaces Through wounds Natural openings

Infection Process by which pathogens establish contact with susceptiblecells or tissues and procure nutrition from them. Successful infections result in symptoms in or on the host Latent infections do not produce symptoms immediately Incubation period is the time between inoculation andappearance of disease symptoms Invasion: movement throughout the host

Colonization Growth and reproduction of the pathogen

Dissemination Further growth sometimes outside the host to new tissues ornew plants By Air By Water By insects, mites, nematodes and other vectors Humans

Overwintering or Oversummering Survival in plant tissues or plant propagative parts In infected plant debris Soil Seeds (parasitic higher plants)

Types of Disease Cycles Monocyclic: single cycle pathogens such as smuts, Polycyclic: more than one generation per growth season;multi cycle pathogens such as downy mildews, late blight ofpotato Polycyclic diseases usually cause explosive epidemics sincethey complete many disease cycles per year and the amountof inoculum is multipled many fold Poletic pathogens: multi-year pathogens; survive in perennialhosts and have as much inoculum at the beginning of theseason as the ended with in the last season. Examples areviruses and phytoplasmas and fungal vascular wilts

Terminology List of terms to study that will be helpful in the cominglectures

plant pathology healthy plant Plant disease symptoms Macroscopic symptoms microscopic symptoms vascular wilt systemic infection hyperplasia hypertrophy

Koch’s Rules Infectious disease symbiosis pathogenicity Obligate parasites Nonobligate parasites Facultative saprophytes Facultative parasites disease triangle disease cycle Monocyclic Polycyclic Poletic pathogens