Molecular detection of latent pathogens in woody plants: current applications and future challenges

Nicola Luchi

CNR – Institute for Sustainable Plant Protection Sesto Fiorentino (Firenze), Italy

Meeting WG 1&2 COST ACTION 1303 - Sustainable control of Grapevine Trunk Diseases

Lubljana, Slovenia, 23 -24 February, 2017 Emerging fungal diseases

Seven pathways driving the emergence of diseases threatening natural and planted forest ecosystems around the world

1. Invasion by alien pathogens; 2. Climate change; 3. Emergence of new virulent and aggressive strains or species; 4. Rise of hybrid fungal species; 5. Latent and cryptic pathogens; 6. Establishment of new associations between vectors and pathogens; 7. Introduciton of new crops and cultivation practices. Invasion by alien species

• Invasive alien species (IAS) are a major threat to biological diversity • Increasing travel, trade, and tourism have facilitated intentional and unintentional movement of species beyond natural geographical barriers

• Trade is one of the main pathways through which IAS can be introduced

Ghelardini et al., (2016) Sudden Oak Dead (SOD)

Phytophthora ramorum Ash Dieback

Hymenoscyphus fraxineus is the causal agent of ash dieback, a severe condition that is currently affecting both Fraxinus excelsior and F. angustifolia in Europe Climate change

Latitude versus year of observation for pest taxonomic groups in the Northern Hemisphere from 1960 onwards.

Bebber et al. (2013) Nature Climate Change, 985-988. Latent pathogens

Some fungal parasites are able to infect host tissue and spend long periods of latency before visible symptoms occur

Trees might be debilitated by drought or other stress inducing the microrganisms to become pathogenic and to produce visible damages Diplodia sapinea

Flowers et al., 2001

Diplodia sapinea

Flowers et al., 2001 Detection of pathogens

Microrganism

Host

DETECTION Disease outbreak

Identify a specific microrganism Disease management Fungal detection (traditional methods)

a) Symptoms and signs

b) Isolation on agarized media

LIMITS of the method: • Difficult to standardise; • Usually they do not give quantitative results; • They are somewhat cumbersome and time–consuming, especially if hundreds of samples have to be analysed; • Some fungal species may be difficult to isolate (masked by endophytic micro-organisms that overgrow them on agar media) Fungal detection (molecular methods)

PCR has proven to be a powerful tool for quantitative analysis of nucleic acids

It is possible to detect a specific microrganism in a mixture of DNA by using reduced quantities of starting material

Main characteristics of this approach: a) Sensitivity b) Rapidity c) Specificity d) Reproducibility Real-time quantitative PCR

New approaches from Clinical science for nucleic acid detection

• Clinical biochemistry: human disease, GMO (Heid et al., 1996; Orlando et al., 1998; Pinzani et al. 2001; Bustin, 2000).

• Forest pathology: - Phaeocryptopus gaeumannii / Douglas-fir (Winton et al., 2002). - Heterobasidion annosum / Norway spruce(Hietala et al., 2003). - Erwinia / Oak (Heuser & Zimmer, 2003). Detection of latent invaders

Similar detection approaches can be used for an early diagnosis of the invasion process in both plants and humans to prevent or reduce the spread of disease qPCR to detect latent pathogen

Biscogniauxia mediterranea -Quercus Specificity

Isolates collected

- B. mediterranea (14)

Related species - B. nummularia (6) - Kretzschmaria deusta (4) - Hypoxylon fragiforme (1) - Xylaria polymorpha (1)

Outgroup - Diplodia mula (4) - Botryosphaeria obtusa (1) - Phythophthora spp. (3) qPCR to detect latent pathogen

Biscogniauxia mediterranea -Quercus Sensitivity and early detection

Range DNA from ascospores [51,7 pg/µl – 6.5x104 pg/µl]

Range DNA from symptomless tissue [0.5 - 440 pg fungal DNA /µg total DNA]

Luchi N., Capre P., Pinzani P., Orlando C., Pazzagli M (2005). Real-me PCR detecon of Biscogniauxia mediterranea in symptomless oak ssue. Le. Appl. Microb. 41, 61-68. qPCR to detect latent pathogen

Biscogniauxia nummularia -Fagus Sensitivity and early detection

The amount of B. nummularia DNA ranged from 250 pg to 8 fg /µg total DNA extracted.

Luchi N., Capre P., Veraino A.M., Vannini A., Pinzani P., Pazzagli M. (2006)- Early detecon of Biscogniauxia nummularia in symptomless European beech (Fagus sylvaca L.) by TaqManTM quantave real-me PCR. Le. Appl.Microb.43,33-38. qPCR to detect airborne inoculum

Certatocyss platani

To assess the spreading of the airborne inoculum during sanitation cuttings qPCR to detect airborne inoculum

Airborne traps

qPCR by using a TaqMan MGB Probe

Luchi et al., 2013. Appl. Environ. Microbiol. 79: 5394-5404 qPCR to detect airborne inoculum

Epidemiology of disease

The inoculum dispersal during the sanitation cuts was evaluated less than 200m

Luchi et al., 2013. Appl. Environ. Microbiol. 79: 5394-5404 qPCR to detect pathogens on insects qPCR detection of Diplodia sapinea on Leptoglossus occidentalis A new insect-fungus association in Mediterranean forests 249

Fig. 2. Diplodia pinea DNA quantification on Leptoglossus occidentalis and pine cones. Insects from laboratory colony were artificially inoculated (CS, Conidial suspension; PCI, Pine cone inoculation) while those not treated were used as Uninoculated control (UC). Samples from forest included insects (VF, Vallombrosa forest; VFW, Vallombrosa forest washed) and pine cones (PC).

4 Discussion Real-time PCR consistently detected D. pinea onLuchi the insects, et al., even 2013. when Appl. these Environ. had been Microbiol. washed. This 79: suggests 5394-5404 that an association had indeed formed between the exotic L. occidentalis and the native D. pinea. Over the last few years, invasive species have been one of the main causes of emerging infective diseases (EIDs) (Bandyopadhyay and Frederiksen 1999; Anderson et al. 2004). Outbreaks of EID occur either when an invading aggressive fungus finds an existing pathway to exploit, or when new vectors invade that are able to transmit a native pathogen more effectively (Bandyopadhyay and Frederiksen 1999). For example, Battisti et al. (1999) found that there was a long-standing association between the native seed bug Orsillus maculatus and the originally exotic fungus, Seiridium cardinale, and showed that the seed bug was now an effective vector of S. cardinale inoculum on cypress cones. In the United States, another close association has been found between the native beetle of elm bark, Hylurgopinus rufipes, and Ophiostoma novo-ulmi, the exotic agent causing Dutch elm disease (Millar et al. 1986). On the other hand, De. valens, a secondary bark beetle in North America, has recently been introduced to China where it has formed an association with native ophiostomatoid fungi, and particularly with Le. procerum (Lu et al. 2009). Leptoglossus occidentalis and D. pinea have the same habitat, offering the same conditions for growth, and this explains why an association has arisen between them. In the present work, this association or interaction was seen when real-time PCR detected D. pinea DNA occurred on both artificially inoculated L. occidentalis and L. occidentalis naturally infected in a forest. Experiments on laboratory-grown insects found that the conidia adhered to the body of the insect, as the highest amount of fungal DNA was detected after conidial inoculation. Comparable amounts of D. pinea DNA were found with other inoculation procedures, as by insects crawling over infected pine cones, or when insects were collected from the forest, where they were probably naturally infected with conidia by coming in contact with infected trees. As D. pinea inoculum can persist for a long time as a saprophyte on the parts of dead trees, such as cones and different coarse woody debris (Santini et al. 2008), the insects may also become infected with D. pinea by crawling on the tree litter. Diplodia pinea was even detected on uninoculated laboratory-grown (control) insects that had only been fed on apparently healthy Austrian pine seedlings and shoots. The explanation for this is probably that the trees that were fed to these insects were already colonized by the fungus in latent form. The fact that nevertheless small amounts of D. pinea DNA were detected in these samples even after they had been washed could be due to the not quite perfect nature of the washing process, which left some conidia adhering to the insect; although it is also possible that when L. occidentalis sucked the young symptomless cones, it ingested some fungal hyphae or fungal DNA fragments occurring on the cones. Although beetles are thought to be the main vectors of plant pathogens in forests, this is the first time a bug was found to act as a possible fungal vector in a forest. However insects, mainly the Heteroptera, have a vital role as vectors of various disease agents such as viruses, bacteria, phytoplasmas and fungi, on cotton, pistachio, citrus and soybean (Mitchell 2004). Heteroptera transmit pathogens mainly by their piercing–sucking mouthpart that they use in feeding (Mitchell 2004). Citrus and pistachio trees are infected with two yeasts, Nematospora coryli and Ashbya gossypii. Infection occurs just at the time when these trees are being fed on by the leaf-footed bug Leptoglossus gonagra F. The bug is clearly associated with these two yeasts, which have also been found in the digestive tract of the insect (Mitchell 2004). Detection of strictly related species

High Resoluon Melng Analysis (HRMA) Early detection of Phytophthora

Compost

Water

Soil Roots

qPCR was able to detect Phytophthora DNA in nursery from different substrate : - symptomless root tissue; - soil of potted healthy plants; - irrigation water; - compost Migliorini et al., (2015) Detection of pathogens by LAMP

LAMP (Loop-mediated isothermal amplification)

Loop-mediated isothermal amplification (LAMP) is a gene amplification method which amplifies DNA with high specificity and efficiency under isothermal conditions

• LAMP method is based on the use of a set of six specially designed primers spanning six to eight distinct sequences on the target DNA.

• The amplification is performed by DNA polymerase, which has strand-displacing activity.

• The whole reaction occurs in a single tube and is divided into two steps: non-cyclic and cyclic. Detection of pathogens by LAMP

Detection on site ! Instrument weight: 2kg Detection of pathogens by LAMP

1. Sampling

2. DNA extracon [2-3 min]

3. LAMP [30 min (16 samples)] Analysis

! Next generation sequencing (NGS) Next generation sequencing (NGS) technologies have impressively accelerated research in biological science during the last years

NGS has been used: a) to study the microbial community in soil, plants, and water (Baptista et al. 2015; Ishoey et al. 2008; Panda et al. 2015), b) to analyses endophyte communities (Cleary et al. 2016) ; c) as a powerful tool for complete genome sequencing in different microbial phytopathogens (Olson et al. 2012). Next generation sequencing (NGS)

However, NGS technologies generate large amounts of sequence data that require:

- bioinformatic expertise; - software for the analysis of the results - ….and TIME before producing definitive results.

Despite the development of these new high-throughput technologies, qPCR and LAMP still represents the gold standard for measurement of nucleic acids and is thus the most appropriate tool for the early detection of latent invaders. Future challenges

1. Prevent the introduction

Early detection before the occurrence of symptoms in the host

Symptomless plant Border inspection Soil

Recently, the advent of Detecon in field Detecon in lab NGS allowed the intercepon of ‘unknown microbial sequences’ that could be related to LAMP qPCR unidenfied pathogens (Laurence et Presence/absence DNA al. 2014). quanficaon Future challenges

Control of estabilished disease

Treatments againts pathogens

Detecon in field Detecon in lab Detecon in lab

LAMP qPCR NGS

Presence/absence DNA Microbial community quanficaon (Symbionts, endophyc fungi) Conclusions

• The introduction and spread of plant pests and diseases among food crops and other plant species, particularly in forestry and horticulture, has significant consequences globally for farmers, policy- makers and the general public.

• The recent initiative by the European Commission to upgrade certain protective measures against plant pests and pathogen is important in reinforcing technical aspects of risk analysis, quarantine and other controls.

• Science and innovation can contribute to improved surveillance, pest and pathogen characterisation, and integrated control options for sustaining plant health; Conclusions

• The need to reduce the time of diagnosis has prompted plant pathologists to move towards more sensitive and rapid methods such as molecular techniques.

• The detection by molecular approaches of latent invaders before the outbreak of disease reduces the impact of plant pathogens.

• These new technological approaches will allow the interception of pathogens before they cause damage to the plant, representing an important tool in the management of plant disease.