Biotic Components of Agricultural Ecosystems · Biotic Components of Agricultural Ecosystems...

Biotic Components of Agricultural

Ecosystems

Valeria Scala

CREA DC

Research Center for Plant Protection and Certification

Roma

Centro di Ricerca per la Difesa e

Certificazione delle Piante

Plant pathology is the study:

of the microrganism and of the enviromental factors thatcause disease in plants;

of the mechanisms by which these factors induce disease in plants

of the methods preventing or controlling disease and reducingthe damage

The challenges for plant pathology are to reduce food losses whileimproving food quality and safe guarding enviroment

A plant becomes diseased when it is continuously disturbed by some causal agent that results in an abnormal physiological process that disrupts the plant’s normal structure, growth, function.

This elicits characteristic pathological conditions or symptoms.

Introduction

Fossil evidence indicates that plants were affected by disease 250 millionyears ago.

The early writings e.g the Bible mention diseases, such as rusts, mildews,blights and blast have caused famine and other drastic changes in theeconomy

Plant disease in more recent times:

late blight of potato in Ireland (1845–60);

powdery and downy mildews of grape in France (1851 and 1878);

coffee rust in Ceylon (starting in the 1870s);

Fusarium wilts of cotton and flax;

southern bacterial wilt of tobacco (early 1900s);

Sigatoka leaf spot and Panama disease of banana in Central America (1900–65);

black stem rust of wheat (1916, 1935, 1953–54);

southern corn leaf blight (1970) in the United States.

Introduction

The Top disease caused by biotic stress

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Biotic Components of Agricultural Ecosystems

Biotic diseases are caused by living

organisms (e.g., fungi, bacteria, and

viruses).

Abiotic diseases are caused by non-

living environmental conditions, (e.g.,

soil, compaction, wind, frost,

soil salt damage, and girdling roots).

Symptoms on a specific part, it does not necessarily mean

the damaged tissue contains the organism causing the

symptoms.

A root rot can cause chlorosis and wilting of stems and

leaves, but the disease causal organism is in the roots.


Symptoms of disease are the plant’s reaction to the causal agent. Plant symptoms include:

Blight (a): A rapid discoloration and death of twigs, foliage, or flowers.

Canker (b): Dead area on bark or stem, often sunken or raised.

Chlorosis: Yellowing

Decline: Progressive decrease in plant vigor.

Gall or gall-like (c) : Abnormal localized swelling or enlargement of plant part. It could be caused by

insects, mites, diseases, or abiotic disorders.

Gummosis: Exudation of gum or sap.

Leaf distortion: The leaf could be twisted, cupped, rolled, or otherwise deformed.

Leaf scorch: Burning along the leaf margin and into the leaf from the margin.

Leaf spot: A spot or lesion on the leaf.

Mosaic: Varying patterns of light and dark plant tissue

Necrosis: Dead tissue – Necrotic areas are also so generic

Stunting: Lack of growth

Wilt: General wilting of the plant or plant part.

Insect feeding injury is also a symptom used in diagnosis, but not a symptom of disease.

a b c


Specific conditions must be present for biotic disease to

develop.

Susceptible host plant, the pathogen (fungi, bacteria,

viruses, etc.), and environmental conditions conducive

to disease development;

these must come together in a given point in time.

These conditions make up what is called the:

Plant Disease Pyramid

Biotic disease cannot occur if one of these pieces is

missing.


Biotic components: microrganism cause abnormal physiological process that disrupts the plant’s normal structure, growth, function.

Morfology and moltiplication of same plant pathogen


Environmental Conditions:

Weather plays a large role in fungal disease development.

Most fungi require free water or specific levels of humidity or

moisture for prolonged periods of time to develop.

Dry climates are not conducive to their survival.

(e.g. the Rocky Mountain region has fewer fungal diseases than many

other parts of the United States due to climatic differences).

Gardens, greenhouse and other microclimates may have conditions ideal for

disease development due to poor air circulation, shade, high humidity, and

high moisture.


A ‘Top 10’ Fungal pathogensbased on scientific/economic importance:

(1) Magnaporthe oryzae; (2) Botrytis cinerea; (3) Puccinia spp.; (4) Fusarium graminearum; (5)

Fusarium oxysporum; (6) Blumeria graminis; (7) Mycosphaerella graminicola; (8)

Colletotrichum spp.; (9) Ustilago maydis; (10) Melampsora lini

Importance and priorities can vary locally across continents and disciplines.

DOI: 10.1111/J.1364-3703.2011.00783.X


M. Oryzae: over one-half of the world’s population relying on rice as the main source of calories, this pathogen

can have devastating effects;

Botrytis cinerea: has an impact in many areas because of the broad host range, causing severe damage,

both pre- and post-harvest;

Puccinia spp.: grouping the three wheat infesting rust diseases have increased in impact with the emergence of

race Ug99, now posing a serious challenge to wheat production;

F. graminearum: significant damage predominantly to cereals and a few non cereal species;

F. oxysporum: a wide host range, with severe losses in crops as diverse as tomato, cotton and banana.

Blumeria graminis and Mycosphaerella graminicola

Colletotrichum spp.: have long served as a model system for hemibiotrophic pathogens, with a short bio-

trophic stage, followed by a switch to tissue ramification and necrotrophic development.

Ustilago maydis and Melampsora lini make up ninth and tenth positions, respectively, with many voters indi-

cating strong scientific rather than economic reasons for their inclusion.

Phakopsora pachyrhizi, the causal agent of Asian soybean rust, has only recently appeared in certain parts of

the world and will potentially increase in importance (Goellner et al., 2010).


Fungi

• Reproduce by spores; the main dispersal mechanism of fungi; remain dormant

until germination conditions are appropriate.

• Many fungi over-winter as fruiting structures embedded in dead plant tissue.

• When a spore comes into contact with a susceptible plant, germinate and enter

the host if the proper environmental conditions are present.

• Hyphae develop from the germinated spore and begin to extract nutrients from

host plant cells.

• Fungi are spread by wind, water, soil, animals, equipment, and in plant material.

• Fungi enter plants through natural openings such as stomata and lenticels and

through wounds from pruning, hail, and other mechanical damage. Fungi can also

produce enzymes that break down the cuticle (the outer protective covering of

plants).

• Fungi cause a variety of symptoms including leaf spots, leaf curling, galls, rots,

wilts, cankers, and stem and root rots.


Magnaporthe oryzae: filamentous ascomycete, the causal agent of rice

blast disease, the most destructive disease of rice worldwide.

All foliar tissues are subject to infection; can lead to complete loss of

grain. Losses of 10%–30% are typical, although regional epidemics can

be devastating; host resistance is the most economically viable and

environmentally sound practice to manage this disease, the fungus

overcomes blast resistance quickly, and cultivars typically become

ineffective within 2–3 years; cause disease on a variety of grasses and

related species, including crops such as barley, wheat. New wheat

strains have emerged in South America.

Knowledge of the biology, genetic diversity and adaptability of this

pathogen is key for the development of novel and durable strategies to

manage devastating fungal diseases. Today, much effort is focused on

characterizing the many avirulence and corresponding rice resistance

genes. The development of genetic markers (MAGGY, MGR583 and

MGR586) provide the molecular tools to assess population diversity and

the evolution of lineages, valuable knowledge for the breeding.

M. oryzae elaborates an appressorium required for infection, indeed,

several effective fungicides, such as tricyclazole, which inhibit

melanization of the appressorium, block host penetration. Considerable

knowledge has been acquired regarding the perception of environmental,

starvation responses, cell signalling pathways, turgor pressure

generation, recycling cellular contents (autophagy) and cell cycle

checkpoints which regulate and orchestrate the development of this

specialized cell


Botrytis cinerea Persoon: Fries [teleomorph Botryotinia fuckeliana (de Bary), grey

mould, can infect more than 200 plant, necrotroph, which co-opts programmed cell

death pathways in the host to achieve infection. The most destructive on mature or

senescent tissues of dicotyledonous hosts. Sometimes remains quiescent for a

considerable time before rotting tissues when the host physiology changes and the

environment is conducive. Infestation can occur in all the way from the seedling stage

until product ripening. Serious damage can occur following the harvest of seemingly

healthy crops. Harvested commodities can be spoiled in the retail chain, during

storage, transport to distant markets or during display at the retailer. Damage occurs

in different stages of the production and retail chain.

In spite of the increasingly effective application of biocontrol, fungicide application

remains the common method to control Botrytis. The average cost for chemical

Botrytis control (all crops, all countries) is about €40/ha (Steiger, 2007). Fungicides

against Botrytis representing 10% of the world fungicide market (UIPP, 2002). Botrytis

control (cultural measures, botryticides, broad-spectrum fungicides, biocontrol) easily

surmount €1 billion/annum.

The wine and table grapes segment represents 50% of the value of the total market

for botryticides, with solanaceous vegetables, cucurbits, strawberries and

ornamentals each making up 5%–9% (Steiger, 2007). Fungicide resistance is an

increasingly problematic issue.

In wine and table grapes, expenses for the control of Botrytis reduce in profit (e.g.

Australia (AUS $52 million/year), Chile (US$ 22.4 million/year)).

In 2002, about 15%–20% of rose and gerbera contained detectable Botrytis infection,

the loss in revenue for rose growers alone was estimated at €1.3 million. Reduction of

the shelf life (fruit) or vase life (flowers) is a serious quality issue.

Botrytis cinerea may occasionally be beneficial!

(Under specific climatic conditions, cause noble rot in grape berries, which are used to

produce sweet wines (Sauternes, Tokaj).


Stem (black) rust (caused by Puccinia graminis f. sp. tritici)(Pgt), stripe (yellow) rust

(P. striiformis f. sp. tritici)(Pst) and leaf (brown) rust (P. triticina)(Pt).

Sporulation, efficient dissemination, pathogenic variability and the widespread

cultivation of wheat, conducive environments, contribute to the destructive potential of

these rusts. Historically, stem rust damage wheat crops, in ancient Rome (rituals

(‘Robigalia’) were performed to save crops from rust).

Obligate, biotrophic basidiomycete, with macrocyclic, heteroecious life cycles

differentiate specialized infection structures, obtain nutrients through feeding

structures, called haustoria, which are situated inside plant cell

Stem rust repeate uredinial stages on common and durum wheat, barley and triticale.

Basidiospores can infect alternative hosts (Berberis vulgaris) (primary inoculum for

wheat and new virulence combinations as a result of sexual re-assortment of genes)

Significant and repeated crop failures caused by rusts occurred in North America

between 1904 and 1962, in Europe and China

Recently, severe and widespread stripe rust epidemics have been ascribed to new

and aggressive races adapted to warmer environments (Hovmøller et al., 2011).

The elucidation of the rust life cycle and the genome sequence are significant steps

towards a better understanding of virulence and breeding for durable resistance. The

specialization in different races has impacted strongly on wheat breeding and

production. Numerous cultivars protected by single genes have become susceptible

to stem rust, often with devastating ‘boom-and-bust’ effects. The race Ug99 of Pgt in

East Africa has renewed stem rust research.

90% of the world’s wheat is susceptible, the Ug99 race group.

Detection, genetic mapping and quantitative trait loci (QTL) conferring resistance are

being made in understanding the molecular basis of pathogenicity in Pgt.

The release and adoption of widely adapted resistant cultivars are essential for future

and effective rust control globally.


The ascomycete Fusarium graminearum (teleomorph Gibberella zeae), is a highly

destructive pathogen of all cereal species. F. graminearum co-exists and co-infects

with other Fusarium species. The greatest economic losses occur when the floral

tissues become infected.

Reduces grain quality and results in mycotoxin contaminated grain.

Worldwide, all the major cereal-growing regions have reported a re-emergence of

Fusarium epidemics. During the post-harvest period, if infected cereal grain is stored or

transported at too high a moisture content, post-harvest growth of the fungus occurs and

mycotoxin levels increase

Mycotoxin is often unsafe for human consumption, animal feed or malting purposes.

In Europe, the USA and other regions, strict upper limits on specific mycotoxin levels

in grain and foods have been imposed [Commission Regulation (EC) 1881/2006;

http://www.scabusa.org].

F. graminearum produces several trichothecene mycotoxins, the most important of

which are deoxynivalenol (DON), acetylated DON derivatives, nivalenol and the

zearalenone. DON inhibits protein translation.

Control of Fusarium floral infections remains problematic. In most cereal species, the

resistance sources identified are only partially effective and are major QTL based

Some azole fungicides are moderately effective, but spray coverage and the timing of

applications remain difficult. Minimizing consecutive cereal crops and ploughing under

any infected residues remain the best methods to reduce disease pressure locally. F.

graminearum infection of non cereal species in the crop rotation is increasingly being

reported, e.g. in soybean and sugar beet.

A considerable phase of symptomless infection exists in which hyphae advance

extracellularly between the living host cells. Host cells only die on intracellular hyphal

invasion, and extensive degradation of plant cell walls is a relatively late process.


Fusarium oxysporum Schlecht. is a ubiquitous soil-borne pathogen that causes

vascular wilt on a wide range of plants. Characteristic disease symptoms

include vascular browning, leaf epinasty, stunting, progressive wilting,

defoliation and plant death (Agrios, 2005). The F. oxysporum species complex

comprises different formae speciales (f. sp.), which infect more than 100

different hosts, provoking severe losses in crops such as

melon, tomato, cotton and banana, among others.

In contrast with the remarkably broad host range at the species level, individual

isolates of F. oxysporum cause disease only on one or a few plant species.

This dichotomy suggest that different isolates of a given forma specialis,

infecting the same host plant, have originated independently during evolution.

F. oxysporum lacks a known sexual cycle, the mechanism through which these

new pathogenic lineages emerged. Recently, analysis of the complete genome

sequence of the tomato pathogenic form F. oxysporum f. sp. lycopersici (Fol)

revealed the presence of lineage-specific (LS) genomic regions, including four

entire chromosomes that are absent from other Fusarium species (such as the

cereal pathogens F. graminearum and F. verticillioides). Transfer of two LS

chromosomes from Fol to a nonpathogenic isolate enabled it to cause disease

on tomato plants. This suggests that horizontal transfer of small chromosomes

could account for the emergence of new pathogenic lineages (Ma et al., 2010).

Dominant plant resistance (R) genes against different races of F. oxyspo-

rum have been identified in several crops These studies led to the identification

of a classical gene-for-gene system with at least three fungal avirulence genes,

some of which can function as both elicitors and suppressors of R gene-based

plant immunity

(A) F. oxysporum microconidium (C) germinating

on the surface of a tomato root. Penetration occurs

by directed growth of the infectious hypha (IH)

towards a natural opening between epidermal root

cells (penetration site indicated by an arrow). (B) F.

oxysporum hypha growing in a xylem vessel of a

tomato root (from Di Pietro et al., 2001)


A Top 10 plant virus list:

(1) Tobacco mosaic virus, (2) Tomato spotted wilt virus, (3) Tomato yellow leaf curl virus, (4) Cucumber mosaic virus, (5)

Potato virus Y, (6) Cauliflower mosaic virus, (7) African cassava mosaic virus,(8) Plum pox virus,(9) Brome mosaic virus,

(10) Potato virus X

with honourable mentions for viruses just missing out on the Top 10, including Citrus tristeza virus, Barley yellow dwarf

virus, Potato leafroll virus and Tomato bushy stunt virus.

The majority of viruses are single-stranded positive-sense RNA viruses, other forms of nucleic acid genomes are double-

stranded DNA (dsDNA), represented by CaMV, with unusual translation strategies, the use of reverse transcription in

replication.

The single-stranded DNA viruses of the Geminiviridae are represented by Tomato yellow leaf curl virus (TYLCV) and

African cassava mosaic virus (ACMV), both having huge economic importance exacerbated by efficient transmission via

whitefly vectors. ACMV cause the annual losses of US$1.9– 2.7 billion, with the cassava disease pandemic in East and

Central Africa.

Tomato spotted wilt virus (TSWV), is transmited by thrips, with negative and ambisense single-stranded RNAs.

Potato virus Y (PVY) and Plum pox virus (PPV), Potyviridae. A worldwide distribution and are efficiently transmitted by

aphids, making them difficult to control. PPV is the most serious viral disease of stone fruit crops, with control measures

costing billions of US dollars over recent years. PVY, also transmitted by aphids, shows further problems created by the

wide range of isolates with highly variable degrees of virulence. PVY also causes significant damage in potato, tobacco,

tomato and pepper


Plant virus


Tobacco mosaic virus (TMV). (A)

Systemic infection of Nicotiana

tabacum cv. Turk plants showing

TMV-associated mosaic. (B)

Necrotic local lesions on N.

tabacum cv. Glurk leaf,

demonstrating Holmes’ N-gene

resistance following inoculation

with TMV.

Symptoms caused by virus:

Different degree of dwarfing

or stunting and reduction of

total yield.

Usually appeared on leaves

but some viruses cause

striking symptoms on the

steam, fruit and root.


Transmission through vegetative

propagation, natural root grafts

and dodder.

Virus trasmission

Mechanical trasmission or sap

trasmissionof plant viruses

Virus trasmission through direct

ontact , handling, seed and pollen


Tomato spotted wilt virustransmitted by thrips with worldwide losses estimated to be in excess of US$1 billion annually by 1994. The continuing

economic importance of TSWV is a result of: (i) its worldwide distribution and wide host range (>800 plant tomato, pepper,

lettuce, peanut and chrysanthemum); (ii) the difficulty in managing the thrip vectors, and hence the virus TSWV causes

variable symptoms

Novel integrated management strategies have been developed for TSWV because the complex vector–virus relationship

and the rapidity of transmission limit the effectiveness of insecticides

The TSWV biology (i) virions contain the viral RNA-dependent RNA polymerase which uses host cell mRNAs to prime viral

transcription via cap-snatching and (ii) thrips can only transmit TSWV if acquired as larvae, although both larvae and adults

are able to transmit. TSWV replicates in its thrip vectors making thrips both vectors and mobile hosts for the virus,

Tomato yellow leaf curl virusTYLCV causes one of the most devastating emerging diseases of

tomato worldwide, is transmitted by the whitefly Bemisia tabaci.

TYLCV has quickly spread from the Eastern Mediterranean Basin

to the entire Middle East, CentralAsia, North andWest Africa,

southeastern Europe, the Caribbean islands, southeastern USA,

Mexico,the Southern Indian Ocean islands and Japan. The rapid

spread of the viral disease is caused by whitefly pressure (Fig. A)

and by high transmission efficacy. A single whitefly is able to

inoculate a plant following a 15-min acquisition period and a 15-min

inoculation period. In the field, inoculation can occur immediately

after transplantation. Infected seedlings will remain stunted and will

not yield fruits (Fig. B). Apart from whiteflies,TYLCV can be

transmitted by grafting, by agroinoculation and by DNA-coated

particle bombardment. It is not seed transmitted.

(A) Numerous whiteflies on a tomato leaf. (B) Top panel,

noninfected tomato plant; bottom panel, typical Tomato

yellow leaf curl virus (TYLCV) disease on a tomato plant.

(C) Infected susceptible (left) and resistant (right) tomato

lines bred for resistance to begomoviruses.


Potato virus YPVY possesses ss(+)RNA genome of approximately 9.7.

A viral genome-linked protein (VPg) is covalently attached to the 5′ end of the RNA and a poly(A)n tail is present at the 3′

end

transmitted by more than 40 aphid species in a nonpersistent manner.

Infects a wide host range mainly within the Solanaceae, and is distributed worldwide.

induce mosaic on tobacco and potato, and leaf drop on potato, are responsible for the partial/total leaf necrosis of infected

hosts

Potato is the fourth most important food crop in the world, with a yield of 315 million tons in 2006 (http://

www.potato2008.org), and a continuous progression (4.5% per year) of the world production of tubers. As a result of a lack

of efficient resistance to PVY isolates inducing leaf/tuber necrotic symptoms in cultivated varieties and the plant-to-plant

transmission of isolates through daughter tubers, the control strategy used to reduce the incidence of PVY is mainly based

on certification of seed production. PVY is also a destructive virus in tobacco crops, causing height reductions and

modifying the chemical composition (e.g. nicotine content). Other crop species affected by PVY include pepper, where

infection rates of 100% have been observed, and tomato, where emerging PVY strains cause serious damage to yields and

fruit quality. Finally, crops with lower economic impacts have also been shown to be strongly affected by PVY (e.g. petunia

in Europe)

PVY on tomato friut (A) and potato leaves (B)AB


Top 10 bacterial plant pathogen list:

The list includes, in rank order: (1) Pseudomonas syringae pathovars; (2) Ralstonia solanacearum; (3) Agrobacterium

tumefaciens; (4) Xanthomonas oryzae pv. oryzae; (5) Xanthomonas campestris pathovars; (6) Xanthomonas axonopodis

pathovars; (7) Erwinia amylovora; (8) Xylella fastidiosa; (9) Dickeya (dadantii and solani); (10) Pectobacterium carotovorum

(and Pectobacterium atrosepticum).

Bacteria garnering honourable mentions for just missing out on the Top 10 include Clavibacter michiganensis

(michiganensis and sepedonicus), Pseudomonas savastanoi and Candidatus Liberibacter asiaticus

Plant pathogenic bacteria and symptoms they cause


Pseudomonas syringae pathovars:

The economic impact of P. syringae is increasing, with a resurgence of old diseases, including bacterial speck of tomato

and the emergence of new infections of importance worldwide, such as bleeding canker of horse-chestnut (pv. aesculi).

Several pathovars cause long-term problems in trees, often through the production of distortions and cankers (e.g.

pathovars savastanoi and morsprunorum).

Ralstonia solanacearumThe most destructive plant pathogenic bacterium worldwide, is composed of a very

large group of strains varying in their geographical origin, host range and pathogenic

behaviour. This heterogeneous group is recognized as a ‘species complex’ which has

been divided into four main phylotypes (phylogenetic grouping of strains).

Infecting 200 plant species, causal agent of potato brown rot, bacterial wilt of tomato,

tobacco, eggplant and some ornamentals, as well as Moko disease of banana.

A soil-borne pathogen infects plants via wounds, root tips or cracks at the sites of

lateral root emergence, colonizes the root cortex, invades the xylem vessels and

reaches the stem and aerial parts of the plant through the vascular system

Can rapidly multiply in the xylem up to very high cell densities, leading to wilting

symptoms and plant death

Responsible for an estimated US$1 billion in losses each year worldwide (Elphinstone,

2005).

The incidence of the disease is particularly dramatic for agriculture in many developing

countries in inter-tropical regions in which is endemic.

Disease management remains limited and is hampered by the faculty of the pathogen

to survive for years in wet soil, water ponds, on plant debris or in asymptomatic weed

hosts, which act as inoculum reservoir


Xanthomonas campestris pathovars:

cause diseases of agronomic importance throughout the world. Among them Xanthomonas campestris pv. campestris

(Xcc), the causal agent of black rot of crucifers that affects all cultivated brassicas, X. campestris pv. vesicatoria (Xcv), now

reclassified as X. euvesicatoria, the causal agent of bacterial spot of pepper and tomato, and X. campestris pv.

malvacearum (Xcm, now X. axonopodis pv. malvacearum), which causes angular leaf spot of cotton. The diseases caused

by these bacteria are particularly severe in regions with a warm and humid climate, although black rot is also economically

important in temperate regions, e.g. in Cornwall and other western areas of the UK. Xcc is also important as a producer of

the EPS xanthan, which is used as a food additive and in the pharmaceutical and oil-drilling industries.

Xcv established the genetic basis of the triggering of disease resistance in pepper, leading to the isolation of genes

specifying avirulence on pepper cultivars containing the Bs1, Bs2 or Bs3 (for bacterial spot) resistance gene.

Plant Diagnostics: Importance

Take a look in signs and symptoms

Quick plant pathogen diagnosis

Perform diagnostic test in specialized

lab

Plant quarantine is a technique for ensuring disease- and pest-free

plants, whereby a plant is isolated while tests are performed to detect

the presence of a problem.


Detection:

Plant protection plays an important role in agriculture for the food quality and quantity.

The diagnosis of plant diseases and the identification of the pathogens are essential

prerequisites for understanding and controlling them.

The “harmonization of phytosanitary regulations and all other areas of official plant

protection action” means the good practices for plant protection and plant material

certification.

Different techniques (microscopy, serology, biochemical, physiological, molecular tools

and culture propagation) are currently used to identify pathogens.

Systems for early detecting diseases can prevent their spread and food losses.

The protection of natural and managed plant systems from alien and emerging indigenous

pests is a strategic socio-economic issue

The cooperation in plant health have been established within the International Plant

Protection Convention (IPPC, https://www.ippc.int/). In particular European and

Mediterrenean Plant Protection Organization (EPPO) is an intergovernmental organization

responsible for cooperation and harmonization in plant protection within the European and

Mediterranean region, under the International Plant Protection Convention (IPPC).

Detection of disease symptoms is the first step that prompt the plant pathologist to apply

diagnostic protocols for the pest management.


Detection:

Among traditional methods the plant symptoms observation

The selective media

The serology-based methods

The molecular tests can be based on hybridization or amplification techniques

To diagnose a new disease the entire array of diagnostic principles must be used

(morphological, biochemical, serological, molecular, etc).

Research, develop innovative methods to achieve results within a shorter time and

sometimes with higher performance

Asymptomatic plants can be a reservoir of the pathogen and the development of diagnostic

methods with improved sensitivity, specificity are helpful for the identification of plant

pathogens, even in the absence of disease symptoms or signs of the causal agent.

The breakthrough for the disease diagnosis is to be found in the spectroscopic and imaging

techniques and the volatile organic metabolites as biomarkers. These techniques are non-

invasive, applicable at point of interest.

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Saggi biologici 6 -36 mesi

ELISA 2 giorni

extraction coating visualization

Molecular hybridization2 giorni

extraction blotting development

PCR 1 - 2 giorni

extraction RT-PCR electrophoresis

Real time -PCR1 giorno

extraction RT-PCR

LAMP-PCR 2-3 ore

extraction RT-PCR

Diagnostic methods

Isolation

Bioassay

Microscopy

ELISA test

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MOLECULAR Test Diagnosi diretta: molecolare

Nucleic acid and probe (base-pairing)

Extraction (DNA-RNA)

Annealing nucleic acid and probe

Detection

C

G

A

C

G

G

U

U

A

C

G

C

U

G

C

C

A

A

U

G

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Extraction RNA or DNA

Protocols

(solventi organici)

Kits

Where (Lab or open field)

Matrix

Sensitivity

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TEST Diagnosi diretta: molecolare

Hybridization AMPLIFICATION

NORTHERN O SOUTHERN BLOTTING

DOT-BLOTTING

TISSUE BLOTTING

PCR

RT-PCR

REAL TIME PCR

NEXT GENERATION SEQUENCING

LAMP

DIGITAL PCR

MICROARRAY

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IBRIDAZIONE MOLECOLARE

membrana di nitrocellulosa

G

C

U

G

C

C

A

A

U

G

C

G

A

C

G

G

U

U

A

C

Dig

AP

development fixing

Pellicola fotografica

PCRDNA o cDNA

3’5’ 5’ 3’

5’ 3’ 3’ 5’

Target is amplified 2n times

Enzyme

Taq

P1 P2

Gel visualization

3’ 5’

5’ 3’

Denaturation

Primer P1

Primer P2 Annealing

Elongation

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HSVd

300 bp

ADFVd

306 bp

PBCVd

315 bp

ASSVd

330 bp

PLMVd

338 bp

CSVd

354 bp

CEVd

370 bp

Results

What’s Wrong With

Agarose Gels?

* Low sensitivity

* Low resolution

* Non-automated

* Size-based discrimination only

* Results are not expressed as numbers

based on personal evaluation

• Ethidium bromide staining is not very quantitative

• End point analysis

ABI: Real-Time PCR vs Traditional PCR (www)

http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

In the frame of Italian roles on phytosanitary

aspects (D.L. n. 214 / August 19/2005)

a national laboratory organization is defined

as follows:

Central

laboratory

Laboratories distributed on the territory

20 Regions

41

National Reference Laboratory

Network of laboratories distributed on

the entire Italian territory

1. To establish official diagnostic protocols

2. To transfer official protocols to the Network

3. To train Network personnel

4. To maintain an official pathogens collection

5. To organize proficiency tests

6. To provide technical-scientific support to the

competent authorities

Tasks:

CREA-DC:

laboratory accreditated

UNI ISO 17025

for

seven test methods

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Sample flow

Phytosanitary inspector is responsable of:

Collection of samples

Pakaging of samples

Shipping of samples

Arrival of samples within 24/48 h from field collection

Pest management

Conventional:Evaluation of pesticides: capability, persistance, residues

Integrated control:Optimization of pesticides use

Reduction of chemical residues

Reduction of environment impact (water, soil, crop, air)

Organic farming:Reduction of the use of plant protection products

Reduction of chemical residues

Reduction of environment impact

Identification of natural products for pathogen control

3

Several pilot research project aimed at increasing the

collaboration among european research groups.

Comparison and validation of detection methods for different pathogens

Ring testing

Assessment of the risk posed by different pathogens

Epidemiological studies on, phytopatogens, reservoir hosts and potential

vectors

45

EPPO standard PM 7/122 (1) –

Guidelines for the organization of interlaboratory

comparisons by plant pest diagnostic laboratories

‘Test performance studies provide added value to

the validation process ‘

EPPO standard PM 7/98 (2) –

Specific requirements for laboratories preparing

accreditation for a plant pest diagnostic activity

[according ISO/IEC STANDARD 17025 (2005)]

‘Guidance on the validation process verification of

performance criteria’

VALIDATION OF DIAGNOSTIC PROTOCOLS

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One of the aims of EPPO is to help its member countries to prevent entry

or spread of dangerous pests (plant quarantine).

EPPO recommends its member countries to: regulate the pests listed as

quarantine pests A1 (pests are absent from the EPPO region) and A2

(pests are locally present in the EPPO region ).

The EPPO A1 and A2 list is reviewed every year by the Working Party on

Phytosanitary Regulations and approved by Council.

https://www.eppo.int/

EPPO is an intergovernmental organization responsible for

European cooperation in plant health

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Xylella fastidiosa and the Olive Quick Decline Syndrome (OQDS)

Grapevine and Citrus were never found

infected

The “Quick decline syndrome” appeared in mid-October 2013

in a restricted area near Gallipoli (Apulia, Italy)

• Detected in olive trees in association with other fungal

pathogens.

• In 2015, a different subspecies of the bacterium was

detected in Corsica on ornamentals (mainly Polygala

myrtifolia).

• Phytosanitary action is being taken to stop the spread

of X. fastidiosa in all countries concerned.

Xylella fastidiosa in olive

By molecular and serological diagnostic tecniques, and electron microscopic

observation the bacterium Xylella fastidiosa was detected in olive infected

tissues

IF

Genetic characterization

CoDiRO is a variant of Xf pauca

identical to a strain infecting

oleander in Costa Rica

Epidemiology

Xf was identified in Philaenus

spumarius captured in diseased

olive groves

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A serious threat to agriculture, the environment and the economy.

About the hosts

An extensive natural host range, which includes many herbaceous and woody plants,

cultivated crops and weeds (over 350 plant species).

many plant species are asymptomatic hosts or may show symptoms several months

after infection, rendering diagnosis and management difficult.

https://www.ippc.int/static/media/uploads/IPPC_factsheet_Xylella_final.pdf


About the vectors

Any xylem sap feeding insect is a potential vector of

Xylella fastidiosa (typical vectors in California and Brazil, Philaenus spumarius in

Italy)

X. fastidiosa:

• a vector-borne pest may lead to the death of the infected plants.

• can induce a range of diseases: Pierce’s disease of grapevine, phony peach disease, olive

quick decline and leaf scorch in almonds, coffee, oleander (depending on the host species and

on the bacterium subspecies (fastidiosa, multiplex, pauca),

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World distribution

• occurs primarily in the Americas.

• recent outbreaks in Southern Italy, Southern France, the Balearic Islands

(Spain) and an isolated finding in Germany constitute

The bacterium has also been reported in Republic of China and Iran.

The (EPPO) distribution (March 2017).

https://www.ippc.int/static/media/uploads/IPPC_factsheet_Xylella_final.pdf

Pathways of entry and spread:

• through the importation of infected plants for planting

• arrived in Europe from Central American countries.

• transmission by xylem sap-feeding insects favours the natural spread of the disease.


In Mediterranean plant species:

e.g. in Nerium oleander and Polygala myrtifolia,

(the natural and urban landscape of Southern Italy,

Corsica and along the Mediterranean coast in France)

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Economic impacts

Xylella fastidiosa subsp. fastidiosa:

• in grapevine causes considerable losses in the USA

• costs California US$ 104 million per year in terms of losses of vines and measures for disease

prevention.

Xylella fastidiosa subsp. pauca:

• in 2007 in Brazil removed around 100 million trees

• control measures against the disease at US$ 120 million per year.

subsp. pauca strain CoDiRO:

• in Apulia region covers approximately 180 000 ha the olive trees in the infected area are

centennial trees.

• the impact of the pathogen on olive is inestimable.

Symptoms depend on hosts and X. fastidiosa strain combinations.

Generally, symptoms include leaf scorching, wilting of the foliage, defoliation, chlorosis or

bronzing along the leaf margin and dwarfing.


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EPPO BulletinVolume 46, Issue 3, pages 463-500, 20 SEP 2016 DOI: 10.1111/epp.12327http://onlinelibrary.wiley.com/doi/10.1111/epp.12327/full#epp12327-fig-0012

X. fastidiosa proliferates only in xylem vessels, in roots,

stems and leaves. The vessels are

ultimately blocked by bacterial aggregates and by

tyloses and gums formed by the plant.


http://onlinelibrary.wiley.com/doi/10.1111/epp.2016.46.issue-3/issuetoc

http://onlinelibrary.wiley.com/doi/10.1111/epp.12327/full#epp12327-fig-0012

26/09/2018

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What can be done:

1. Preventive and management measures:

• prevention and/or containment measures such as the use of

‘healthy’ propagating materials,

• early surveillance and detection of the pathogen

• the destruction of infected plants

• vectors control strategies

Agronomical practices may be effective in containing the

disease, such as the pruning of symptomatic wigs/branches in

citrus showing Citrus variegated chlorosis at early stages.


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