Managing aboveground pests: vectors of pathogens: Part I February 17, 2021 Lecture Overview

Part I (this week): • Vector-borne disease concepts • Introduction to Disease Vectors • • Biology • Vector-borne diseases of citrus • Viral Pathogens

Part II (next week): • Vector-borne diseases of citrus • Bacterial Pathogens • Case studies: • Citrus Variegated Chlorosis and Citrus greening disease Learning objectives:

• Learn the mechanisms of transmission of plant pathogens. • Learn the terminology of insect transmission. • Think about the interaction of the plant, pathogen, and vector. Think about factors affecting transmission by . • Know the common groups of that transmit pathogens The most destructive plant diseases are vector-borne • Plant pathologists have a different point of view about vector- borne diseases than entomologists. • Plant pathology view: the most destructive plant pathogens are those which are transmitted by vectors • Disrupt the pathogen multiplication in plant host. • Entomology view: One insect can cause economic damage by transmitting a pathogen to plant. • Controlling the insect vector is the key to stopping the disease. • Disruption of pathogen acquisition by the insect from the plant may lead to total control of the disease. Economic Injury Level (EIL) - the lowest pest population level that will cause economic damage or the critical population density where the loss caused by the pest equals in monetary value to the cost of management. Economic Threshold or Action Threshold (ET) - the point at which management actions should be taken to prevent an increasing pest population from exceeding the economic injury level. The ET always represents a pest density or level of pest damage lower than the EIL. Plant disease epidemics

• In special cases, where pests serve as vectors of plant or diseases, the economic threshold (ET) is zero. A single pest attack may cause the death of a plant or animal. • Among many factors affecting the epidemic of plant disease, vectors are the most important factor. • The vector is responsible for disease spread by visiting plants and feeding on specific tissue where the pathogen exists and multiplies. Plant disease epidemics

• Can result in large loss of crop yields or decimate entire plant species (e.g. Dutch elm disease) • Approximately 30-40% of Pathogen damage/loss due to plant diseases (Vector) due to direct or indirect effects of transmission and facilitation of pathogens by insects • Three elements needed for disease to occur (‘disease Susceptible Conducive triangle’) host environment • When a pathogen requires a vector to be spread, the vector must be plentiful and active for an epidemic to occur Disease Triangle • Not shown: time (latent period) for disease development Plant disease epidemics Mechanisms of insect transmission of plant pathogens • The insect transmission of plant pathogens is a complex process that includes specific interactions among the plant host, the insect vector, and the pathogen. • The insect needs to acquire the pathogen from an infected plant and inoculate the pathogen into a healthy plant in order to complete the transmission process. Vectors

• Vector: an organism that does not necessary cause disease by itself, but spreads the infection by conveying pathogens from one host to another. Vectors of plant disease • Mites • Insects • Nematodes Types of vectors:

• Mechanical vectors: • pathogen does not multiply within mechanical vectors • physically transport the pathogen from host to host • Biological vectors: • pathogen must multiply within a biological vector before the biological vector can transmit the pathogen. Vector/Pathogen Relationships

• The ability of a pathogen to survive and remain infective in or on a vector species is a critical factor in disease transmission • Passive • Vectors feeding in or walking through an infected plant area carry pathogen on their body • Generally an inefficient mechanism for disease transmission • Example: Citrus canker • Active (mechanical or biological) àtrue VECTORS Transmission types

• Transmission types are classified depending on pathogen behavior within the vector. • Including: Circulation, Propagation, Persistence • We will cover all of these types in greater detail. Transmission: A complex process

Four major components. 1. Pathogen behavior within the Pathogen vector: 1- Persistence 2- (Vector) Circulation 3- Propagation 2. Vector feeding behavior: 1- Specific feeding site 2- specific feeding tissue 3- Susceptible Conducive background matching host 3. Tritrophic specific environment interaction: 1- Plant-vector interactions 2- Pathogen vector interactions 3- Vector-plant interactions Disease Triangle 4. Factors affecting the transmission efficiency: Environmental Circulation Circulative pathogens Propagation Main mechanisms of pathogen transmission by insects Understanding how vectors transmit pathogens increases knowledge of their epidemiologies and needed for development of disease management • Non-persistent • Semi-persistent • Persistent Transmission types

• Non-persistent transmission- These pathogens (viruses) are acquired from infected host plants through the probing behavior of the insect in very short periods of time. • The insect then transmits the virus by probing on a healthy plant. • Mouthpart-borne (foregut or stylets) and non- circulative. Main mechanisms of pathogen transmission by insects Non-persistent: • Retained by the vector mainly in the stylet (food canal) • Does not circulate in body • Vector feeds on infected plant and carries pathogen on mouthparts to a new plant during subsequent feeding • No latent period • Not retained through molt Transmission types

• Semi-persistent transmission- Viruses are acquired and transmitted by feeding rather than by probing. • The longer the insect feeds, the greater the rate of transmission. • The virus is retained for a few days and varies with the virus/vector combination. Modes of plant pathogen transmission by vectors Semi-persistent (foregut-borne) transmission: • Transmitted by the vector from a few hours to a few days post acquisition • Short AAP, a function of the time it takes to reach the plant vascular tissue • Short retention time (24-48 h) • Lost at molt • Retained mainly in the foregut • No latent period • Many whitefly-transmitted viruses Foregut-borne pathogens adhere to the food canal of the stylets or higher up in the foregut

Managing aboveground pests Transmission types

• Circulative non-propagative transmission: Viruses are acquired and transmitted by the feeding rather than the probing behavior of the insect vector. • 8- to 12-hour latent period for the virus to get from the gut to the salivary glands • longer feeding periods increase the chance of acquisition. Circulative non-propagative transmission • No replication in vector (non-propagative) • Latent period may be a few hours to a few days • Retained in vector for days or weeks (hemocoel, organs) • For many persistently transmitted plant pathogens, particularly in those infecting non-vegetative crops (e.g. citrus), insect transmission is obligatory for the pathogen Transmission types

• Circulative propagative pathogens include viruses and bacteria Circulative propagative transmission • Propagative: • Replication occurs in the vector • Long AAP • Latent period may be days or weeks before the vector is able to transmit the pathogen • Pathogen often retained for the lifespan of the vector (hemocoel, organs) • Often transmission to progeny occurs through infection of embryo or germ cells in the female (transovarial transmission) • Spiroplasma citri (Citrus stubborn disease) Non-circulative propagative transmission • The only example for this type of transmission is Xylella fastidiosa. • Foregut limited- attaches and forms a bacterial biofilm. That is different!

• The bacterium is located in the vector foregut (ciberium and pre-ciberium) • Nymphs lose the bacterium after molting while adults maintain the bacterium for their whole life. • Nymphs and adults transmit X. fastidiosa • No latent period No transmission after molting • No transovarial transmission Transmission parameters

• Transmission parameters • Acquisition Access Period (AAP): the period of time given for the vector to acquire the pathogen from plant. • Inoculation Access Period (IAP): the period of time given for the vector to transmit (inoculate) the pathogen to plant. • Latent Period: the time between acquiring the pathogen by the vector and the ability to transmit it to plant. Pathogen Transmission

• Transmission: transfer of a pathogen from an infectious source to a susceptible host, includes acquisition and inoculation • Acquisition access period (AAP): time required for a vector to ingest a pathogen from an infected plant host

• Image: https://www.sciencedirect.co m/science/article/abs/pii/S2 214574516300657 Pathogen Transmission

• Latent period: time between the acquisition and inoculation of a pathogen by the vector • Inoculation time period or inoculation access period (IAP): time required for a vector to transfer a pathogen to a susceptible plant Transmission parameters

• Transovarial Transmission: transmission of pathogen from female parent to offspring through the ovaries. • Sexual Transmission: transmission of pathogen between infected and uninfected vector adults during courtship. Viruliferous: vector that is capable of transmitting the virus to numerous plants within its lifetime once it acquired the virus. Insect Body Insect body: Barriers to persistent transmission 1. midgut infection 2. dissemination (including midgut escape and salivary gland infection) 3. salivary gland escape 4. transovarial transmission (vertical transmission)

Passage of persistent viruses through different organs in their insect vectors requires specific interactions between a pathogen and vector components Types of vector-borne plant pathogens • Viruses • Bacteria • Mollicutes: Spiroplasmas and phytoplasmas • Proteobacteria Virus vs. Bacteria

• Not living organisms • Living organisms • Subcellular • Unicellular (no true nucleus) • DNA or RNA, not both • Genetic material is DNA • Systemic infection • Localized infection by bacteria • Shape (helical, cubal, complex or the toxins they produce viral shape) • Shape (coccus, bacillus, spiral) • Replicate in host • Replicate by itself by binary fission https://3.bp.blogspot.com/- S7hL1IRWzdA/WPh_v- 83leI/AAAAAAAAJIY/FmitiwtqoE0PmxGCegcqv egcqMaaVdzPQCLcB/s1600/Mitosis%2Bvs%2B Meiosis.png Vector-borne citrus virus and virus-like diseases Case Studies 1. Citrus Vein enation: It is widely distributed, but causes little economic damage. 2. Citrus Leprosis: brevipalpus mites 3. (CTV): aphids ; the most famous and economically important citrus virus • Citrus chlorotic dwarf: bayberry whitefly (Parabemesia myricae) • Satsuma dwarf: soil-borne vector? • Citrus ringspot virus: aphids? • Citrus yellow mosaic: mealybugs • Etc. Case Study 1: Citrus vein enation (CVEV)

• Virus-like (possible leutovirus) • Symptomless in most commercial cultivars, but may cause vein enation and woody gall (lime, rough lemon) • Persistently transmitted by aphids spp. • citricida, Myzus persicae, and Aphis gossypii • 2-3 d latent period in the vector Citrus vein enation (CVEV)

USA: California CVEV: Woody gall CVEV: Vein enation Case Study 2: Citrus Leprosis

• Problem on sweet orange varieties • Caused by virus that is not systemic in plant • Primarily a disease of oranges, but may affect mandarins • Distribution: highly important disease of citrus in South, Central, and North America (recently Panama) Citrus Leprosis: Causal agent

Causal agent • Associated with two distinct rhabdovirus-like viruses, one residing in the cytoplasm and the other in the nucleus of infected cells • Virus particles can be observed by electron microscopy of lesions from naturally infected citrus and from mechanically inoculated Chenopodium quinoa (universal virus host) plants Citrus Leprosis: Symptoms

• Chlorotic lesions on citrus leaves, fruit, and twigs • Chlorotic lesions eventually may become necrotic in the center • Flat or depressed lesions on fruit with concentric patterns and gumming • Abscission of leaves and fruit and twig dieback due to extensive lesion development • Yield reduction, decline and eventual death of citrus trees Leprosis: Leaf lesions Leprosis: Stem lesions

Leprosis: Fruit lesions Leprosis: Fruit drop

Citrus Leprosis: Vector

• Vector: Brevipalpus mites (Family: Tenuipalpidae- flat or false spider mites) • Several species are reported to be vectors: B. californicus, B. obovatus and B. phoenicis • Only B. phoenicis has been confirmed to be an effective vector in experimental transmission studies • According to Knorr (1959) B. californicus was vector in Fl. No voucher specimens available. Citrus Leprosis: Transmission

• Highest transmission efficiency occurs in larval stage rather than at nymphal or adult stages • No transovarial transmission • each newly hatched larvae needs to feed on infected plant tissues in order to acquire the virus • Evidence for persistent propagative transmission: • Transstadial transmission • Retain ability to transmit the pathogen after being held on unsusceptible host plants • Infective throughout lifespan Citrus Leprosis: the Florida situation • Prior to 1925, leprosis was a serious disease of citrus in Florida • Reports from 1906-1968: leprosis nearly destroyed Florida citus industry • It is not known why leprosis disappeared in Florida • Improved mite control? à Sulfur (overuse can kill crops, bad for beneficials) • Replanting in new areas • Miticide costs on Brazilian citrus exceed 90 million US dollars per year (Omoto 2000) • We have the vector(s) • B. phoenicis has more than 1000 plant hosts Citrus Leprosis: the Florida situation Prevention • When Brevipalpus mites colonize fruits, particularly those infected by scab lesions, their development is better than when reared on leaves. Hence, the longer the mites remain on the fruits, the better the conditions for their population to increase • Harvest an important aspect of disease management • Quarantine the importation of both citrus and woody ornamental plants that serve as hosts for Brevipalpus phoenicis (Geijskes), B. californicus (Banks), and B. obovatus Donnadieu (Acari: Tenuipalpidae) from countries where citrus leprosis occurs. Case study 3: Citrus Tristeza Virus (CTV) • RNA virus • Virus strains or isolates may vary from mild to severe, causing little damage to severe decline • Sour orange rootstocks are highly susceptible, while others are resistant • Mild isolates widespread in Florida, widely disseminated by aphids and in budwood • Virus may be present in trees on resistant rootstocks and may provide a reservoir of virus that aphids can be transmitted to susceptible rootstocks CTV: Symptoms

Mild isolates • Trees reduced in size, vigor, and fruit yields Severe isolates • Stem-pitting strains: may cause stem-pitting in limes, grapefruit, and sweet orange • Not present in Florida • Severe strains: wilt, followed by quick decline and ultimate tree death in several weeks • Wilting, dieback, "quick decline," leaf chlorosis and curling, heavy fruit set, honeycombing, • Decline-inducing strains of the virus may be present in trees on resistant rootstocks and may provide a reservoir of virus that aphids can be transmitted to susceptible rootstocks CTV: Stem-pitting Global distribution of the Citrus tristeza virus CTV

Vector, T. citricida CTV: The Florida situation

• Decline of many citrus varieties on sour orange rootstock • Solution: change rootstock • CTV isolates with no detectable symptoms • Vehicles for expressing foreign genes in host phloem CTV: Vectors

• Brown citrus , • introduced in Florida in 1995 • the most efficient vector of the virus • transmission efficiency may vary between virus isolates • establishment of T. citricida in Florida may have caused rapid spread of decline-inducing isolates of CTV • Cotton or melon aphid, Aphis gossypii • less efficient , but effective vector • Green citrus or spirea aphid, , and the black citrus aphid, • relatively inefficient vectors of CTV in Florida CTV: Vectors

Brown Citrus Aphid, Toxoptera citricida

Cotton or melon aphid, Aphis gossypii CTV: Vectors

Green citrus or spirea aphid, Aphis spiraecola

Black citrus aphid, Toxoptera aurantii CTV: Transmission

• Semi-persistent transmission • Short or no latent period • Aphids acquire the virus within minutes of feeding on an infected plant and transmit it to healthy plants within minutes • Aphids lose the ability to transmit the virus within 24-48 hours after acquiring the virus from an infected plant • Chemical or biological control of the aphid is unlikely to reduce the disease spread in the field since acquisition and transmission of the virus by the aphid usually occurs before the aphid is killed by an • Aphid control in nurseries and on budwood source trees important for reducing infection rates CTV: Virus

• Filamentous particles (2000 x 11 nm size) • Genome includes 12 open reading frames, encoding 19 protein products • In most trees, virus exists as a complex of different strains, or sub-isolates • Composition of complex determines symptoms, can be identified using molecular genetic techniques, such as reverse- transcription polymerase chain reaction (RT-PCR) What is RT-PCR? What is RT-PCR?

• Reverse-transcritption PCR • Viral diagnostic assay • Not Real-time PCR! Example of Florida CTV isolate complex: Florida isolate FS627 • Isolate includes decline strain of CTV • Contains mixture of T36, T30, and VT strains (genotypes) • Strains differ in their ability to by transmitted by Aphis gossypii Roy and Brlansky, 2009 • Parent FS627 isolate: T36 most abundant • But, aphids transmitted T30 more frequently, changing the population dynamic of CTV mixtures Roy and Brlansky, 2009

Parent FS627 isolate: T36 most abundant

T36 FS627 Roy and Brlansky, 2009 BUT, aphids transmitted T30 more frequently, changing the population dynamic of CTV mixtures Aphid-transmitted CTV T30 FS627 T36 T30

Why is this important? Roy and Brlansky, 2009 BUT, aphids transmitted T30 more frequently, changing the population dynamic of CTV mixtures Aphid-transmitted CTV T30 FS627 T36 T30

The presence of hidden severe isolates with mild isolates found with Australian, Floridian, and Corsican isolates: single- BCA transmission used as a tool for separation Example of Florida CTV isolate complex: Florida isolate FS627

• Isolate FS687 contains mixture of T36 and T30 isolates • T36: decline- inducing, low aphid transmission • T30: non-decline inducing, high aphid transmission Vector-transmitted citrus pathogens: prognosis

• Most of the major Insect orders are represented, but some orders predominate. • Homoptera have contributed the largest number of species Vector-transmitted citrus pathogens: prognosis

• Immigration is constant • Underscores importance of surveillance for detection of new vectors, pathogens Next Week: Vector-borne plant pathogens and Case Studies • Viruses • Bacteria and bacteria-like microorganisms • Mollicutes: Spiroplasmas and phytoplasmas • Proteobacteria Any questions?