Mycosphaerella Spp Causing Leafspot Disease on Bananas Advanced Methods Supplement

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT

MYCOSPHAERELLA SPP. CAUSING LEAFSPOT DISEASE ON BANANAS

ADVANCED METHODS SUPPLEMENT

Edited by

J. Henderson (UQ/DPI&F Tree Pathology Centre ) K. Grice (Centre for Tropical Agriculture, DPI&F)

Oc to ber 2008 1

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT

MYCOSPHAERELLA SPP. CAUSING

LEAFSPOT DISEASE ON BANANAS

ADVANCED METHODS SUPPLEMENT

OCTOBER 2008

Contributors

Juliane Henderson (UQ/DPI&F)

Kathy Grice (DPI&F)

Marie‐Françoise Zapater (CIRAD)

Françoise Careel (CIRAD)

The PCR primers and reaction conditions described in Protocol 4 of this document are pending publication and thus must be held in confidence until such time they appear in the public domain. Please also ensure these parameters do not appear in material including conference oral presentations, poster and funding body reports. No part of this document, including images, may be reproduced without permission of the authors.

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT

INTRODUCTION ...... 5 1.0 IDENTIFICATION OF MYCOSPHAERELLA SPP. PATHOGENS ON BANANA (IN SITU) USING LIGHT MICROSCOPY...... 8 1.1 AIM...... 8 1.2 METHODOLOGY...... 8 1.3 MATERIALS AND REAGENTS ...... 8 1.4 PROCEDURE ...... 9 Lesion Selection...... 9 Glass Slide Preparation ...... 10 Decolourisation ...... 10 Lesion Observation ...... 10 1.5 PATHOGEN IDENTIFICATION ...... 12

2.0 PRODUCTION OF CULTURES FROM ASCOSPORES OR DIRECT ISOLATION FROM LEAF MATERIAL TO IDENTIFY MYCOSPHAERELLA SPP. ON BANANA...... 19 2.1 AIM...... 19 2.2 METHODOLOGY...... 19 2.3 MATERIALS AND REAGENTS ...... 19 2.3.1 RECIPES ...... 20 2.4 PROCEDURE ...... 20 Ascospore Ejection ...... 20 Ascospore Culture ...... 22 Direct Isolation ...... 23 2.5 MORPHOLOGICAL CHARACTERISTICS OF THE PERFECT FORM OF MYCOSPHAERELLA SPP...... 24

3.0 IN VITRO PRODUCTION OF MYCOSPHAERELLA SPP ASEXUAL STRUCTURES FOR IDENTIFICATION USING LIGHT MICROSCOPY...... 27 3.1 AIM...... 27 3.2 METHODOLOGY...... 27 3.3 MATERIALS AND REAGENTS ...... 27 3.3.1 RECIPES ...... 28 3.4 PROCEDURE ...... 28 Culture and sporulation...... 28 Cotton blue staining ...... 29

4.0 DETECTION AND IDENTIFICATION OF MYCOSPHAERELLA SPP. ON BANANA USING REAL-TIME PCR ...... 30 4.1 AIM...... 30 4.2 METHODOLOGY...... 30 Real-Time PCR Background...... 30 TaqMan® Hybridisation Probe Assays ...... 30 Mycosphaerella spp on Banana Real-time PCR Diagnostic Assay Design...... 32 Internal Amplification Control ...... 36 Enzyme Mastermix ...... 36

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT

4.3 MATERIALS AND REAGENTS ...... 37 Equipment...... 37 Reagents and Suppliers...... 37 4.4 PROCEDURE ...... 39 Preparation and storage of real-time PCR stocks...... 39 Notes on Real-time PCR set up...... 39 Real-time PCR Set Up Protocol...... 40 Real-time PCR Thermal Cycling Protocol...... 43 4.5 RESULTS INTERPRETATION ...... 44 Control Assays...... 44 Mycosphaerella spp on Banana Probe Assays...... 45 Calling a Sample Result...... 46

ACKNOWLEDGEMENT...... 48

REFERENCES ...... 48

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT

INTRODUCTION

This supplement appends the original Sigatoka Leafspot Disease Manual (2006). The purpose of this supplement is to describe advanced methods for the diagnosis of Mycosphaerella fijiensis (black Sigatoka, BS), Mycosphaerella musicola (yellow Sigatoka, YS) and Mycosphaerella eumusae (Eumusae leafspot disease, ELS).

Real-time PCR assays are now routinely used to support the morphological diagnosis of Sigatoka leafspot diseases in Australia. Recently, updated in situ and in vitro techniques were acquired during a laboratory exchange with French scientists Drs Jean Carlier, Marie- Françoise Zapater and Françoise Carreel at the French Agricultural Research Centre for International Development (CIRAD) in Montpellier. In return, Australian-developed, molecular diagnostic assays for black Sigatoka, yellow Sigatoka and Eumusae leafspot disease were transferred to CIRAD for use in routine surveillance of bananas grown in French offshore regions (French Guiana, Guadaloupe, Martinique, New Caledonia and Réunion).

Over 20 species of Mycosphaerella have now been shown to occur on banana (Arzanlou et al., 2008). Three of these species are involved in the Sigatoka disease complex (M. fijiensis, M. musicola and M. eumusae) and surveillance for these pathogens is carried out in Australia.

A fourth species, Mycosphaerella musae, causes speckle disease which is common in the sub-tropical growing regions of South-east Queensland and New South Wales. Mycosphaerella speckle presents with distinct symptoms making diagnosis straightforward. Light-brown or tan-coloured irregular blotches on the lower leaf surface (appearing as smoky patches on the upper surfaces) tending towards dark purple/black, irregularly-shaped, speckled areas are readily found on both leaf surfaces. More information on the pathogen, including symptom images can be found in Jones (2000). Since Mycosphaerella speckle is readily identifiable by symptomology, other methods for diagnosis (ie. morphology and molecular assays) have not been pursued. Importantly, M. musae is significantly different at the sequence level from the pathogens of the Sigatoka leafspot disease complex and therefore, its presence does not interfere with molecular detection assays. This has been confirmed by cross-specificity testing.

The strategy for diagnosis of Sigatoka leafspot disease in banana leaf material suspected of infection, is highly dependent on the type and quality of leaf material available. The following flow diagram illustrates the diagnostic process. Traditional methods of identification rely on light microscopy of fungal structures (in situ, Protocol 1) or following fungal culture (in vitro, Protocols 2 and 3). Molecular diagnosis (real-time PCR, Protocol 4) is used when fungal

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT structures are absent or not able to be cultured, and to confirm diagnosis made by traditional methods. Points to note:

The ELS molecular diagnostic assay in its current format is undergoing final field validation (as at October 2008) and as such, should not be used stand alone in diagnosis. Until this validation has been completed, confirmation of diagnosis as ELS must also be made using traditional (microscopy) techniques.

In Protocol 1, diagnosis is based on structures of the asexual (conidial) stage and therefore the anamorph names are presented for Mycosphaerella fijiensis (anamorph Pseudocercospora fijiensis), Mycosphaerella musicola (anamorph Pseudocercospora musae) and Mycosphaerella eumusae (anamorph Pseudocercospora eumusae). Differentiation based on structures of the sexual (ascospore) stage is not possible as they appear identical.

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT

Diagnosis of Mycosphaerella spp. on banana

Classic Diagnostics Molecular Diagnostics (Light microscopy) (Real-time PCR)

In situ In vitro

Observe directly and/or decolourise Fungal isolation foliar lesions to detect asexual structures PROTOCOL 2

PROTOCOL 1 Isolation of Mycosphaerella spp. Identification of pathogen of banana Mycosphaerella spp. from (i) ascospores or pathogens of banana (ii) direct isolation from using light microscopy leaf tissue

Sporulation of the Sequence-specific pathogen to produce amplification of DNA asexual structures for identification PROTOCOL 4

PROTOCOL 3 Detection and identification of Identification of Mycosphaerella spp. Mycosphaerella spp. using real-time PCR pathogen of banana by light microscopy

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

1.0 IDENTIFICATION OF MYCOSPHAERELLA SPP. PATHOGENS ON BANANA (IN SITU) USING LIGHT MICROSCOPY

1.1 AIM

To identify the banana cercosporoids M. fijiensis (imperfect form Pseudocercospora fijiensis), M. musicola (imperfect form Pseudocercospora musae) and M. eumusae (imperfect form Pseudocercospora eumusae) by microscopic observation of asexual structures (imperfect form) present on or within foliar lesions.

1.2 METHODOLOGY

Leafspot symptoms are scanned using a dissecting microscope, if fungal structures are easily seen a glass slide can be prepared and structures observed at a higher magnification using a compound microscope. This technique is very quick and the specimen can be preserved indefinitely. Alternatively, leaf tissue with suspect lesions is decolourised to allow visualisation of fungal structures in situ. Tissues prepared by decolourisation last approximately one month.

1.3 MATERIALS AND REAGENTS

Falcon® tubes (50mL) Scissors Scalpel blades and handle Glass microscope slides and coverslips Dissecting and compound microscopes Cotton blue staining solution 10% KOH solution

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

1.4 PROCEDURE

LESION SELECTION

Mycosphaerella fijiensis (anamorph Pseudocercospora fijiensis)

Cut 15mm square pieces of leaf tissue (Figure 1.1) containing stage 2 lesions (black lesions of approximately 1cm in length and 2mm in width)

Figure 1.1 Example of Stage 2 lesions required for identification of M. fijiensis

Mycosphaerella musicola (anamorph Pseudocercospora musae) and Mycosphaerella eumusae (anamorph Pseudocercospora eumusae)

Cut 15mm square pieces of leaf tissue containing advanced symptoms (stage 4) as shown in Figure 1.2. Select black/brown lesions before centres turn grey, approximately 1cm in length and 4mm in width

Figure 1.2 Example of Stage 4 lesions required for identification of M. musicola or M. eumusae 9

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

GLASS SLIDE PREPARATION

Using a dissecting microscope, scan lesions as shown in Figures 1.1 and 1.2 for fungal structures. If the leaf material is suspected of being infected by M. fijiensis, ensure the underneath surface of the lesions are assessed, however the top side should be surveyed if M. musicola or M. eumusae are thought to be the cause. If fungal structures are observed (Figure 1.3) these can be picked off the leaf surface and placed in a droplet of cotton blue staining solution as described in Section 3.4. Microscope preparations should then be observed using a compound microscope looking for structures as described in Section 1.5 Pathogen Identification.

Figure 1.3 Tufts of M. musicola conidia as seen on the upper surface of stage 4 lesions

DECOLOURISATION

To decolourise/clear the leaf tissue, place samples as shown in Figures 1.1 and 1.2 into Falcon® tubes containing 10% KOH. Leave overnight (minimum) at room temperature, then rinse the samples well with distilled water.

LESION OBSERVATION

Mycosphaerella fijiensis (anamorph Pseudocercospora fijiensis)

Place the decolourised leaf tissue with the lower surface (banana leaf underside) facing up on a glass microscope slide. Place a few drops of cotton blue staining solution on the surface of the tissue, then cover with a glass coverslip. Seal with clear nail polish if long term storage is required. Ensure the parenchyma cells observed are not palisade (Figure 1.4). Search the slide for conidiophores and conidia as described in Section 1.5 Pathogen Identification. 10

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

Figure 1.4 Underside of decolourised banana leaf tissue (Note the non-palisade parenchyma cells)

Mycosphaerella musicola (anamorph Pseudocercospora musae) and Mycosphaerella eumusae (anamorph Pseudocercospora eumusae)

Place the decolourised leaf tissue with the upper surface (banana leaf topside) facing up on a glass microscope slide. Place a few drops of cotton blue staining solution on the surface of the tissue, then cover with a glass coverslip. Seal with clear nail polish if long term storage is required. Ensure the parenchyma cells observed appear palisade (picket fence like structure, Figure 1.5). Search the slide for conidiophores and conidia as described in Section 1.5 Pathogen Identification.

Figure 1.5 Upperside of decolourised banana leaf tissue (Note the picket fence type, palisade parenchyma cells)

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

1.5 PATHOGEN IDENTIFICATION

Morphological characteristics of Mycosphaerella fijiensis ( anamorph Pseudocercospora fijiensis)

Conidiophores in situ

Conidiophores are pale to medium oliveaeous brown and emerge from stomata (Figure 1.6) within the lesion either singly or in diverging fascicles of 2-8 stalks, predominantly on the lower leaf surface. There are no sporodochia and the stroma is sporadic. Conidiophores can be straight or bent, often with several geniculations, sometimes with a basal swelling up to 8µm diameter, 0-5 septate, 16.5-62.5µm long, 4-7 µm wide, usually slightly narrower at the tip. One or more scars are present near the tip of the conidiophore indicating the attachment point for the conidia as shown in Figures 1.7a and b (this is an important diagnostic characteristic of the organism).

Figure 1.6 Diagram of vertical sections through the lower epidermis of a banana leaf demonstrating the origin of M. fijiensis conidiophores and their emergence from stomata (Meredith, 1969)

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

Figure 1.7a and b M. fijiensis conidiophores emerging from stomata (arrows indicate ‘scars’, a diagnostic feature of M. fijiensis).

Conidia in situ and in vitro

Conidia are not quite colourless but pale green or olivaceous, obclavate to cylindro- obclavate, 1-10 septate (5-7), straight or slightly curved with a truncate or rounded base and a visible slightly thickened hilum. Conidia are 30-132µm long and 2.5-5µm wide at the base which is the broadest point and tapering at the apex as shown in Figures 1.8, 1.9a and 1.9b.

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

Figure 1.8 Diagram of M. fijiensis conidia from diseased leaf material (Meredith, 1969)

Figure 1.9a M. fijiensis conidia in vitro (Note the basal scar on the conidia) b. Conidia of M. fijiensis in situ (Note the basal scars on the conidia)

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

Morphological characteristics of Mycosphaerella musicola (anamorph Pseudocercospora musae)

Conidiophores in situ

Sporodochia (mass of tightly aligned conidiophores on a dark stroma) develop in the substomatal air chamber and the conidiophores grow through the stoma pore in a tuft-like fashion (Figures 1.10, 1.11a and 1.11b). As more conidiophores emerge (predominantly on the upper surface), sporodochia become erumpent, guard cells become disrupted and the adjacent epidermis is pushed back. Conidiophores are pale to very pale olivaceous brown, paler towards the apex, straight or slightly curved, rarely branched, aseptate, not shouldered or geniculated, narrow towards the apex and without conidial scars. Conidiophores are bottle-shaped, with rounded or nearly truncate apices and measure 5-25µm in length.

Figure 1.10 Diagram of M. musicola sporodochia (Meredith, 1970)

a b

Figure 1.11a & b M. musicola sporodochia in situ (with and without conidia attached)

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

Conidia in situ and in vitro

Conidia are borne terminally and singly on the conidiophore. Their appearance is pale to very pale olivaceous brown, smooth, straight or variously curved, almost perfectly cylindrical to obclavate-cylindrical as illustrated in Figures 1.12, 1.13a and 1.13b. Apex is obtuse or subobtuse and the basal cell is shortly attenuate and has no thickened basal hilum. Conidia are usually 2-5 septate or more and measure 10-80µm in length x 2-6µm in width.

Figure 1.12 Diagram of M. musicola conidia (Meredith, 1970)

a b

Figure 1.13 a & b In vitro conidia of M. musicola taken with 40x objective (Note there is no basal scarring present on the conidia)

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

Morphological characteristics of Mycosphaerella eumusae (anamorph Pseudocercospora eumusae)

Conidiophores in situ

Sporodochia of M. eumusae and M. musicola develop in a similar fashion and predominantly develop on the upper leaf surface. Young or immature sporodochia are observed to be subepidermal and substomatal (Figure 1.14a and 1.14b), however the former is characterised by producing an epiphyllous sporodochia. Conidiophores aggregate in dense fascicles arising from the upper cells of a brown stroma up to 70µm wide as illustrated in Figure 1.15. Conidiophores subcylindrical, smooth, hyaline or pale brown toward the base, 0-3 septate, straight to geniculate-sinuous, unbranched or branched below, 10-25µm in length x 3-5µm in width.

Figure 1.14a Sporodochia of M. eumusae (in situ) embedded in the leaf tissue (Note conidiophores are not obvious as seen for M. fijiensis and M. musicola) b. Sporodochia of M. eumusae (in situ) with conidia protruding through the leaf epidermis

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 1

Figure 1.15 Diagram of both asexual and sexual M. eumusae fungal structures (Crous and Mourichon, 2003)

Conidia in situ and in vitro

Conidia are solitary, subhyaline to pale olivaceous, thick-walled, smooth, subcylindrical, apex obtuse, base subtruncate, straight to variously curved, 3-8 septate, 30-50µm in length x 2.5-3µm width with an inconspicuous hila (Figures 1.16a and 1.16b).

a b

Figure 1.16a Conidia of M. eumusae (in situ) erupting from the leaf surface b. Conidia of M. eumusae in vitro

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 2

2.0 PRODUCTION OF CULTURES FROM ASCOSPORES OR DIRECT ISOLATION FROM LEAF MATERIAL TO IDENTIFY MYCOSPHAERELLA SPP. ON BANANA

2.1 AIM

Cultures of Mycosphaerella spp. can be derived from ascospores (sexual stage) or from direct leaf isolations. These cultures can be used to produce conidia (asexual stage) for species identification.

2.2 METHODOLOGY

The ascospore method requires dried leaf tissue containing necrotic lesions which are wetted causing the perithecia to rupture and eject their ascospores. Germinated ascospores are removed to PDA and subsequently subcultured onto sporulation medium to produce conidia for identification (Protocol 3). To produce cultures directly from leaf material, early disease development stages are required. Material is surface sterilised and small pieces of tissue are excised and plated onto PDA media. To induce sporulation refer to Protocol 3, as previously mentioned.

2.3 MATERIALS AND REAGENTS

Scalpel blades and handle 3% water agar (90mm diameter Petri dishes) (see 2.3.1 RECIPES) PDA (90mm diameter Petri dishes) (see 2.3.1 RECIPES) 1% and 2% sodium hypochlorite solution Sterile distilled water Sterile blotting paper Tweezers 25ºC incubator with 12hr day/12hr night light cycle Dissecting microscope with light source underneath stage Bunsen burner Parafilm®

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 2

2.3.1 RECIPES

3% Water Agar Bacto Agar (DIFCO) 30 g

H2O 1000 ml

PDA Potato Dextrose Agar (DIFCO) 39 g

H2O 1000 ml

Media Preparation

Autoclave culture media, cool to 50-55ºC and aseptically add: Penicillin G at dosage rate 100U/mL Streptomycin sulfate at dosage rate 100µg/mL Pour into sterile 90mm Petri dishes Plates containing antibiotics are stable for 1-2 weeks when stored at 4ºC

2.4 PROCEDURE

ASCOSPORE EJECTION

1. Select leaf tissue with necrotic lesions (most likely to contain ascospores), particularly those with grey centres which under increased magnification contain small black spots (perithecia). Figure 2.1 illustrates the progressive enlargement of necrotic lesions as seen using a dissecting microscope under increasing magnification.

2. Banana leaf samples must be very dry. If necessary, leave tissue at room temperature 2-3 days to dry. Dried tissue up to 2 months old can be used for ascospore ejection

3. Cut leaf tissue containing coalescing, necrotic lesions into 4cm square pieces

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 2

Figure 2.1 Progressive enlargements of necrotic material required for ascospore ejection

4. Immerse leaf tissue into a 2% solution of sodium hypochlorite for 2 min. This is to rid/reduce any surface fungi that may be present. Rinse in sterile distilled water to remove excess sodium hypochlorite

5. Place leaf tissue into beakers that contain sterile distilled water for 20-30 min

6. Invert a 3% water agar plate and arrange the tissue pieces in the lid. Ensure the upper leaf surface is closest to the agar surface (see Figure 2.2) as this is where most perithecia are produced. Outline the location of the tissue pieces on the bottom of the agar plate in permanent pen to assist location of germinated ascospores following incubation (see Figure 2.3)

7. Incubate at 25ºC for 24-48 hours in a 12hr day/ 12hr night cycle

Water agar Surface with most perithecia facing agar

Dried banana leaf tissue

Figure 2.2 Diagram of ascospore ejection plate set-up

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 2

Figure 2.3 Marking the position of leaf pieces on the base of the water agar plates to assist the location of germinated ascospores following incubation

ASCOSPORE CULTURE

1. Look for germinated ascospores using a dissecting microscope fitted with an illuminated stage plate. Focus efforts on the regions defined on the plate base in permanent pen

2. Select only ascospores which have one or two terminal germtubes (Figure 2.4)

3. Using a sterile scalpel blade, dissect a small agar square containing the germinated ascospore and transfer the agar square onto a fresh PDA plate (Figure 2.5). Between 8 - 10 ascospores can be isolated onto a PDA plate

4. Seal the plate with Parafilm® and incubate at 25ºC for 10 days

5. Ascospore cultures are transferred to V8 sporulation media for the production of conidia (see Protocol 3)

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 2

Figure 2.4 Germinated ascospore (Note germtubes emerging bilaterally)

Figure 2.5 Agar plug containing germinated ascospore plated onto fresh PDA (Image courtesy of CIRAD)

DIRECT ISOLATION

1. Select leaf material containing Stage 2 lesions of suspected M. musicola (Figure 2.6a), or Stage 2-3 for M. fijiensis or M. eumusae (Figure 2.6b) and cut into 1-2cm squares

a b

Figure 2.6a and 2.6b Lesion stage required to direct isolate the Mycosphaerella spp. organisms from leaf tissue (M. musicola and M. fijiensis/M. eumusae) 23

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 2

2. Immerse tissue into a beaker containing 1% sodium hypochlorite for 1 min, remove and blot dry using sterile blotting paper

3. Using a sterile scalpel blade, make incisions on either side of the lesion, then horizontally as shown in Figure 2.7, taking care not to cut right through the leaf piece

Figure 2.7 The dotted lines illustrate where the scalpel incisions are made in relation to the lesion.

4. Excise epidermal pieces of tissue (approximately 2mm square) and plate onto PDA

5. Seal the plate with Parafilm® and incubate at 25ºC for 10 days

6. Transfer small portions of the culture to V8 sporulation media for the production of conidia (see Protocol 3)

2.5 MORPHOLOGICAL CHARACTERISTICS OF THE PERFECT FORM OF MYCOSPHAERELLA SPP.

Combined description from Brun (1963) and Jones (2000).

The two structures of the perfect form (perithecia and ascospores) have identical morphology in the three major Mycosphaerella spp that occur on banana. Therefore the sexual stage on its own cannot be used as a diagnostic feature.

• Perithecia are mostly globose with a diameter ranging between 47-85µm, immersed in the leaf tissue with protruding ostioles, and are found on both leaf surfaces

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 2

although more abundant on the upper. The wall of the perithecium is dark brown with three or more layers of polygonal-shaped cells. • Asci (containing ascospores) are bitunicate, obclavate and without paraphyses. Size ranges from 30-40µm in length and 8-12µm in width. Between 10 and 27 asci are found per perithecium for M. musicola. • Ascospores (8 per asci) are hyaline, biserate and ellipsoidal with length 14-18µm and width 3-4µm.

Figure 2.8 illustrates a perithecia which has been manually crushed to release the asci. Figure 2.9 is a diagrammatic cross-section through a perithecium, showing asci and ascospores. Figures 2.10a and 2.10b are ascospores of Mycosphaerella fijiensis which have germinated after 48 hours incubation on water agar

Figure 2.8 Manually crushed perithecia releasing asci (Image courtesy of CIRAD)

asci

ascospores

Figure 2.9 Cross-section of a Mycosphaerella musicola perithecium (Brun, 1963)

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 2

a b

Figures 2.10a and 2.10b Germinated ascospores of M. fijiensis (Note germtubes emerging from either end) (Images courtesy of CIRAD)

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 3

3.0 IN VITRO PRODUCTION OF MYCOSPHAERELLA SPP ASEXUAL STRUCTURES FOR IDENTIFICATION USING LIGHT MICROSCOPY

3.1 AIM

To produce asexual structures (conidia) in vitro for the identification of cercosporoids causing leaf spot diseases of banana.

3.2 METHODOLOGY

Small pieces of mycelia are cultured on modified V8 medium to induce conidial sporulation. Conidia are then harvested, stained and observed using light microscopy to identify the causal organism.

3.3 MATERIALS AND REAGENTS

Ascospore cultures (from Protocol 2) Bunsen burner or spirit flame to sterilise instruments Scalpel blades and handles V8 Sporulation media (see 3.3.1 RECIPES) Parafilm® Incubation chamber (20ºC, continuous cool white fluorescent light) Cotton blue stain (see 3.3.1 RECIPES) Glass microscope slides and coverslips Clear nail polish to seal slide preparations Compound microscope with 10 - 40x objectives and/or oil immersion lens

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 3

3.3.1 RECIPES

V8 Sporulation Media V8 Vegetable Juice 100 ml

CaCO3 0.2g

H2O 900 ml Adjust to pH 6 with 1M NaOH Agar 20 g

Media Preparation

Autoclave culture media, cool to 50-55ºC and aseptically add: Penicillin G at dosage rate 100U/mL Streptomycin sulfate at dosage rate 100µg/mL Pour into sterile 90mm Petri Dishes Plates containing antibiotics are stable for 1 - 2 weeks when stored at 4ºC

Cotton Blue Staining Solution

Mix an equal volume of glycerol and lactic acid Dissolve 0.5g of cotton blue per 100mL of glycerol/lactic acid mix Filter using a 5µm Millipore membrane into a clean Schott bottle

3.4 PROCEDURE

CULTURE AND SPORULATION

1. Using a flame-sterilised scalpel blade, excise small pieces of mycelium from the active edge of ascospore cultures produced using Protocol 2 2. Place mycelium pieces onto V8 sporulation medium (3-4 pieces per plate). Flame sterilise scalpel blade between excisions 3. Seal plates with Parafilm® and incubate at 20°C for 10 to 14 days under 60 µmoles m-2 s- 1 of continuous and cool-white fluorescent light

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS ADVANCED METHODS SUPPLEMENT - PROTOCOL 3

COTTON BLUE STAINING

1. Place a drop of cotton blue solution on a clean, glass microscope slide

2. Scrape cultures grown on V8 sporulation medium with a sterilised scalpel blade to pick up mycelium and conidia (Figure 3.1)

Figure 3.1 Scraping conidia and mycelium from an ascospore-derived culture

3. Suspend scraped conidia directly into the cotton blue droplet, emulsify culture in stain using coverslip to assist if necessary (Figure 3.2)

Figure 3.2 Emulsifying conidia in cotton blue stain

4. Place coverslip over the droplet of staining solution, taking care to prevent bubbles forming in stain solution. Coverslip may be sealed permanently using clear nail polish

Observe using 10 – 40x objectives or oil immersion lens. See Section 1.5 Pathogen Identification in Protocol 1 for morphological characteristics of M. fijiensis, M. musicola and M. eumusae conidia

MYCOSPHAERELLA SPP CAUSING LEAFSPOT DISEASE ON BANANAS

ADVANCED METHODS SUPPLEMENT - PROTOCOL 4

4.0 DETECTION AND IDENTIFICATION OF MYCOSPHAERELLA SPP. ON BANANA USING REAL‐TIME PCR

4.1 AIM

To confirm the identification of Mycosphaerella spp. on banana, especially where fungal structures used for morphological identification (Protocols 1-3) are absent

4.2 METHODOLOGY

REAL‐TIME PCR BACKGROUND

Real-time PCR uses fluorescent dye molecules to detect PCR product accumulation during the assay. Data is collected in ‘real-time’ during the run, and this strategy eliminates the need for post-PCR product handling techniques such as gel electrophoresis. The advantages of real-time PCR include faster processing time, increased sensitivity and reduced cross-contamination due to the closed tube format. Importantly, the technique is also quantitative. The amount of fluorescence generated during the exponential stage of the PCR reaction, can be used to accurately quantify the amount of starting template in a sample. This is in contrast to conventional PCR methods which analyse the end products of the PCR assay when reaction components are exhausted.

The most common real-time PCR formats either incorporate intercalating dyes into PCR products during amplification (eg. SYBR® Green 1), or utilise specific probes labelled with reporter dye molecules (eg. TaqMan® hybridisation probes, Scorpion® probes, Molecular Beacons®). For diagnostic targets, the highest levels of specificity and sensitivity are achieved using probe technology.

TAQMAN® HYBRIDISATION PROBE ASSAYS

TaqMan® hybridisation probe assays incorporate specific forward and reverse primers as well as a specific internal hybridisation probe. This design confers two- level specificity to the target. The probe, which is dual-labelled with a reporter fluorophore at the 5’end and a non-fluorescing quencher molecule in close proximity at the 3’end, operates under the principles of fluorescence resonance energy transfer

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(FRET). This means that while the probe is intact, the fluorescence emitted by the 5’ reporter dye is effectively quenched by the 3’ quencher dye. Upon cleavage of the probe, the two dyes are spatially separated allowing the 5’ reporter dye to fluoresce. Taqman® MGB probes contain a minor groove binder (MBG) moiety at the 3’end which greatly increases the stability and specificity of probe hybridisation. Using MGB technology, probe sequences up to half the length of those of conventional TaqMan® probes, may be designed. This is very useful when working with difficult target sequences.

The probe is designed to hybridise at a temperature 10ºC higher than the specific primers, ensuring it is bound to the target template before primer extension begins. Following annealing of the primers, extension using Taq DNA polymerase is carried out. During extension, the 5’→3’ exonuclease activity of Taq polymerase cleaves the probe, spatially separating the fluorophores and emitting detectable fluorescence. This fluorescence increases exponentially as amplification progresses, with one fluorophore molecule being released for each template strand produced during the PCR. The fluorescence data generated during the run is collected using a sequence detection system (real-time PCR thermocycler), and analysed using inbuilt software to quantify the starting amount of template in the sample. Figure 4.1 outlines the TaqMan® hybridisation probe assay.

Figure 4.1 Diagram of TaqMan® hybridisation probe assay chemistry (Source: www3.appliedbiosystems.com) 31

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MYCOSPHAERELLA SPP ON BANANA REAL‐TIME PCR DIAGNOSTIC ASSAY DESIGN

The real-time PCR diagnostic assays for black Sigatoka (Mycosphaerella fijiensis) and yellow Sigatoka (Mycosphaerella musicola) are based on the TaqMan® MGB probe assay format. MGB technology was necessary to avoid difficult sequence regions through the design of shorter probes. The assay for Eumusae leaf spot disease (Mycosphaerella eumusae) uses TaqMan probe technology without MGB modification.

Mycosphaerella musicola has been found to exist as a group of many different subspecies; based on ITS sequence data, our studies have revealed seven distinct clades worldwide. In Australia, two distinct populations of Mycosphaerella musicola predominate (designated Clade 1 and Clade 6). To ensure reliable detection of both of these clades, two independent probes are employed in the diagnostic screening.

As for gel-based Sigatoka PCR, the real-time assays are designed to amplify specific sections of the ribosomal DNA. Using this region allows good species differentiation as well as good sensitivity as ribosomal DNA is present in high copy numbers in the pathogen genome. The probe target sites for M. fijiensis and M. musicola are located in the internal transcribed spacer region 1 (ITS1). These assays have specific forward primers in the ITS1 (M. fijiensis = MF14F; M. musicola = MM14F) and share a common reverse primer located in the conserved 5.8S gene region (MYCO200R). The product sizes for the yellow Sigatoka and black Sigatoka assays are 154bp and 165bp respectively. The TaqMan® MGB probe for yellow Sigatoka Clade 1 (MMVIC) possesses a VIC label while both the black Sigatoka (MFFAM) and yellow Sigatoka Clade 6 (MM2) probes possess a FAM label. Both probes have a non-fluorescent quencher (NFQ) which contributes no fluorescence to the assay, thus decreasing the background. The probe target site for M. eumusae is located in the ITS2, and the assay utilises a forward primer located in the conserved 5.8S gene region (ELS_GEN2_F1) and a highly specific reverse primer in the ITS2 (ELS_GEN2_R1). A regular TaqMan® hybridisation probe (with no MGB modification), specific to all known ELS sequences and containing a FAM reporter dye and Black Hole Quencher 1 (BHQ1) confers a second level of specificity to the 165bp product. The oligonucleotide sequences for the probes and primers are listed in Table 4.1. Locations of the pathogen primers and probes on the ribosomal gene region are shown in Figure 4.2.

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Target Oligo Type Oligo Name Location Sequence 5’→3’ M. fijiensis Forward primer MF14F ITS1 CACGCCCGACCTCCAA M. fijiensis Reverse primer MYCO200R 5.8S ATCGATGCCAGAACCAAGAGAT M. fijiensis TaqMan® MGB Probe MF-FAM ITS1 FAM-AGGCCGTCTAAACACT-MGBNFQ M. musicola Forward primer MM14F ITS1 CACCCCCGACCTCCAAC M. musicola Reverse primer MYCO200R 5.8S ATCGATGCCAGAACCAAGAGAT M. musicola (Clade1) TaqMan® MGB Probe MMVIC ITS1 VIC-AGGTCTCCTTAACACTGCAT-MGBNFQ M. musicola (Clade 6) TaqMan® MGB Probe MM2 ITS1 FAM-TCTCCCTCACACTGCAT-MGBNFQ M. eumusae Forward primer ELS_GEN2_F1 5.8S CGTCATTTCACCACTCAAG M. eumusae Reverse primer ELS-GEN2_R1 ITS2 GAGGTCAACCTTTCAATAAAGA M. eumusae TaqMan® Probe ELS_GEN2_as ITS2 FAM-AAGAATCCACAACGCTAGGAGACGGA-BHQ1

Table 4.1 Mycosphaerella spp real-time PCR assay oligonucleotide primer and probe sequences

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1 11 21 31 41 51 M.musicola TGCGGAGGGATCATTACCGAGTGAGGGCTCACCCCCGACCTCCAACCCTTTGTGAACCAC M.fijiensis TGCGGAGGGATCATTACCGAGTGAGGGCTCACGCCCGACCTCCAACCCTTTGTGAACCAC M.eumusae TGCGGAGGGATCATTACTGAGTGAGGGCTCACGCCCGACCTCCAACCCTCTGTGAACCAC M.musae TGCGGAGGGATCATTACCGAGCGAGGGCTCCGGCCCGACCTCCAACCCTTTGTGAATCAA

61 71 81 91 101 111 M.musicola A-CCTGTTGCTTCGGGGGCGACCCTGCCGGCGAACTTGTCGCCGGGCGCCCCCGGAGGTC M.fijiensis AACTTGTTGCTTCGGGGGCGACC-TGCCG------TCGGCGGGCGCCCCCGGAGGCC M.eumusae ACTTTGTTGCTTCGGGGGCGACCCTGCCGGCGAACTCGTCGCCGGGCGCCCCCGGAGGTC M.musae AACCTGTTGCTTCGGGGGCGACCCTGCCGT-----TCGCGGCGCGGCGCCCCCGGGGGAA

121 131 141 151 161 171 M.musicola TCCTTAACACTGCATC--TCTGCGTCGGAGTTCCAAACAAATCGGACAAAACTTTCAACA M.fijiensis GTCTAAACACTGCATC—-TTTGCGTCGGAGTTTAAAACAAATCGAACAAAACTTTCAACA M.eumusae TTCTAAACACTGCATC—-TCTGCGTCGGAGTTCAAAACAAATCGAACAAAACTTTCAACA M.musae ATC-AAACACTGCGTCAATTTGGGTCGGAGTACTTGTTAAT--AAACAAAACTTTCAACA

181 191 201 211 221 231 M.musicola ACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAA M.fijiensis ACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAA M.eumusae ACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAA M.musae ACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAA

241 251 261 271 281 291 M.musicola TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTTTGGCATTCCGA M.fijiensis TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTTTGGTATTCCGA M.eumusae TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTTTGGTATTCCGA M.musae TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCGTGGTATTCCGC

301 311 321 331 341 351 M.musicola AGGGCATGCCTGTTCGAGCGTCATTTCACCACTCAAGCCTAGCTTGGTATTGGGCGCCGC M.fijiensis AGGGCATGCCTGTTCGAGCGTCATTTCACCACTCAAGCCTGGCTTGGTATTGGGCGTCGC M.eumusae AGGGCATGCCTGTTCGAGCGTCATTTCACCACTCAAGCCTAGCTTGGTATTGGGCGTCGC M.musae GGGGCATGCCTGTTCGAGCGTCATTTCACCACTCGAGTCTGACTCGGTATTGGGCGCCGC

361 371 381 391 401 411 M.musicola GGTGCTCCGCGCGCCCCAAAGTCTCCCGGCTGAGCCGTCCGTCTCTAAGCGTTGTGGATT M.fijiensis GGTTCTTCGCGCGCCTTAAAGTCT-CCGGCTGAGCTGTCCGTCTCTAAGCGTTGTGGATC M.eumusae GGTGTTCCGCGCGCCTTAAAGTCTTCCGGCTGAGCTGTCCGTCTCCTAGCGTTGTGGATT M.musae GTTTCGATGCGCGCCTTAAAGTTT-CCGGCTGGACCGTCCGTCTCCGAGCGTTGTGGCAT

421 431 441 451 461 471 M.musicola TTTCAGTTCGCTCCGGAGCGCGGGTGGCCGCGGCC-GTTAAATCTT----CAAAGGTTGA M.fijiensis TTTCAATTCGCTTCGGAGTGCGGGTGGCCGCGGCC-GTTAAATCTTTATTCAAAGGTTGA M.eumusae CTTCAATTCGCTTCGGAGTGCGGGCGGCCGCGGCC-GTTAAATCTTTATTGAAAGGTTGA M.musae CTGT—-CTCGCTAGGGAGTCGCGGAGGGCGTTGGCCGTTAAACACCCCATCAAAGGTTGA

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481 491 501 511 521 531 M.musicola CCTCGGATCA-GGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAG------M.fijiensis CCTCGGATCAGGTARGGGATACCCGCTGAACTTAAGCAT------M.eumusae CCTCGGATCA-GGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAAAGGG M.musae CCTCGGATCA-GGTAGGGATACCCGCTGAACTTAAGCATATCAATAA------

Figure 4.2 Diagnostic oligonucleotide primer and probe sites. Forward primers for each pathogen are shaded green, reverse primers are shaded blue and TaqMan® hybridisation probes are shaded yellow. M. musicola Clade 6 probe, which is shorter than the Clade 1 probe, is underlined.

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INTERNAL AMPLIFICATION CONTROL

To avoid potential false negative results, which may arise from the DNA extraction method or the presence of Taq DNA polymerase inhibitors, an internal amplification control PCR assay has been developed to the host DNA. The assay is designed to detect the putative, single-copy gene banana pectate lyase (MWPL-1), and has been validated against 59 varieties covering all genotypes (AA, AAA, AAAB, AB, AAB, ABB, BB) as well as 18 other known and unknown Musa species. The assay has been applied to gel-based PCR (where it produces a 180bp PCR product), and through inclusion of a fluorescent probe, is a highly reliable control for real-time PCR. The primers and probes for the internal amplification control are listed in Table 4.2.

Oligo Type Oligo Name Sequence 5’→3’ Forward primer MWPL1-110 GTCCGCGACCCTGAATTAGTAGT Reverse primer MWPL1-197 CGGTGCCGCATGACAAG TaqMan® Probe MWPL1-ROX ROX-CGCCGCGACACGTTCAAGCTT-BHQ2

Table 4.2 Banana internal amplification control primer and probe sequences

ENZYME MASTERMIX

The advantages of using a mastermix rather than individual components lies in quality control, reproducibility and ease of set-up. RealMasterMix Probe® (5’Prime) has been selected due to its superior performance in the Sigatoka real-time PCR assay when compared to other commercially available PCR mastermixes. RealMasterMix Probe® outperformed the other mastermixes in areas including fluorescence intensity, signal to noise ratio and assay reproducibility. RealMasterMix Probe® contains an enzyme ‘inhibitor’ which prevents non-specific amplification at temperatures lower than 40ºC. The mix is also self-adjusting for Mg2+ concentration through the presence of an agent which weakly chelates excess Mg2+ ions in the reaction. RealMasterMix Probe® is supplied at a 2.5X stock concentration and can be stored either at -20ºC for its shelf life, or at 4ºC for up to 3 months.

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Note: It is important to use the correct enzyme mastermix for the real-time machine being used as some platforms require the incorporation of ROX dye to normalise for possible variations between the wells. RealMasterMix Probe® is available (+/- ROX) and ordering details for both are included in this protocol. When using machines that require ROX for normalisation, an alternative fluorescent reporter dye must be used for the internal control probe.

4.3 MATERIALS AND REAGENTS

The list following is tailored to suit the Corbett Research RotorGene® (RG6000). Amend where necessary to suit the real-time platform available in the testing laboratory.

EQUIPMENT

Pipettes: P2, P20, P200, P1000 Aerosol resistant/filter tips (racked, sterilised, certified DNase/RNase free) Sterile 1.5mL microfuge tubes Plastic rack to hold 1.5mL/2.0mL tubes 0.1mL tubes and caps for RG6000 (Corbett Research P/N 3001-002) Alloy PCR block to fit 0.1mL tubes (Corbett Research P/N 3001-008 or 3001- 028) Fine-tipped permanent ink marker Vortex Microcentrifuge or Capsulefuge to suit 1.5mL and 2.0mL tubes RotorGene® Thermal Cycler RG6000 (Corbett Research) PPE including powder-free, latex gloves

REAGENTS AND SUPPLIERS

RealMasterMix Probe® 2.5X 200Rxns (5Prime P/N 2200700) or RealMasterMix Probe® ROX 2.5X 200Rxns (5Prime P/N 2200720)

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Sterile water (Millipore® or injectable grade) PCR primers (HPLC-purified grade) TaqMan® MGB probe or TaqMan® probe

Item Product Number Company Information TaqMan MGB Probe 4316034 Applied Biosystems (6000pmole) 52 Rocco Drive (M. fijiensis, M. musicola, M. Scoresby VIC 3179 musicola Clade 6) AUSTRALIA T: 1800 801 644 F:03 9730 8798 E: [email protected] NOTE: 6-8 week delivery Dual-labelled BHQ1 Probes DLO-FB1-5 Biosearch Technologies, Inc. 100nmol 81 Digital Drive (ELS, internal amplification Novato, CA 94949-5750 control) USA T: 1-415-883-8400 F: 1-415-883-8488 E: [email protected] RealMasterMix Probe® 2.5X P/N 2200700 Quantum Scientific Pty Ltd (200 rxns) 1/31 Archimedes Place Murrarie QLD 4172 RealMasterMix Probe® ROX P/N 2200720 T: 1800 777 168 2.5X (200 rxns) F: 1800 625 547 E: [email protected] Oligonucleotide primers GeneWorks Pty Ltd PO Box 299 Hindmarsh SA 5007 T: 1800 882 555 E: [email protected]

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4.4 PROCEDURE

PREPARATION AND STORAGE OF REAL‐TIME PCR STOCKS

Reagent Preparation/Storage Details

Sterile water • Injectable water (eg Pfizer) is ideal

• Store in sterile vials at room temperature

RealMasterMix Probe® 2.5X or • Store for shelf life in a constant RealMasterMix Probe® ROX 2.5X (200 temperature freezer at -20ºC rxns) • Store at 4ºC for 3 months

Primer stocks • Primers can be ordered in solution at 100µM

• Prepare a 10µM working solution by diluting 1:10 in sterile water and store aliquots at -20ºC

• Do not dilute all stock as primers are more stable at higher concentrations

Probe stocks • Probes can be ordered in solution at 100µM

• Prepare a 10µM working solution by diluting 1:10 in sterile water and store aliquots in the dark at -20ºC

• Probe efficiency reduces dramatically after approximately 6 freeze/thaws, therefore minimise thaws where possible

NOTES ON REAL‐TIME PCR SET UP

Real-time PCR is extremely sensitive and therefore extra precautions should be taken to prevent the likelihood of contamination. • PCR set-up should be carried out in a designated, “PCR-clean” area, using dedicated equipment. A PCR-clean laboratory coat should be worn. No movement of equipment or reagents should occur between PCR-clean and general laboratory areas, excepting prepared reactions which may travel one-way to the template addition area. Backwards movement of

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racks between the template addition area to the PCR preparation area must be avoided. • Internal amplification control PCR reactions must be set up in parallel with diagnostic samples for quality control. • It is recommended to only set-up one PCR experiment per day. PCR set- up should never directly follow handling of templates. • Reagents, including RealMasterMix Probe®, should be thoroughly thawed, mixed and pulse-centrifuged to collect contents at the bottom of the tube. • Preparation of mastermixes is recommended to standardise reagents across the tubes and minimises pipetting errors. When preparing mastermixes, include extra tubes for positive (1 tube) and negative (3 tubes) PCR controls. Three negative controls are included for use by the software algorithm. At least duplicates are run per banana sample. Also, allow extra tubes (usually 10% of the number being tested, ie. 20 tubes + 2 extra) to account for losses due to errors caused by tip retention during aliquotting.

REAL‐TIME PCR SET UP PROTOCOL

1. In a sterile 1.5mL microfuge tube, prepare mastermixes for each of the assays (yellow Sigatoka Clades 1 and 6, black Sigatoka and Eumusae Leaf Spot) by adding reagents in the order they appear Table 4.3. Reagent amounts are for a single reaction (final volume 20µL), allowing for addition of 2µl of DNA template, or 2µL of sterile water in the negative PCR control tubes.

Note: At the same time as you are setting up the diagnostic assays, ensure that a separate internal control assay is set up in parallel for each sample being screened.

2. Aliquot 18µL of mastermix to 0.1mL tubes (positioned in alloy block). Add 2µL of sterile water to three negative controls and fit lids. To ensure a reliable control against contamination during mastermix preparation, the negative control tubes must remain closed from this point.

3. Mark the lids (where the machine permits) with the tube order and transfer the tubes to the template addition area. Where possible, racks should not be removed from the PCR clean area. If this cannot be avoided, the tubes should be transferred to another rack before template addition and the ‘template-free’ rack returned to the PCR-clean laboratory immediately.

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4. Using filter tips, add 2µL template DNA (from a 1:10 dilution of extract) to individual PCR tubes. The use of filter tips at template addition ensures the source of DNA being tested and prevents cross-contamination from pipette barrels. As soon as practical, close the lids. Add 2µL of positive control DNA to the last tube, making sure that all sample tubes with unknowns are closed first.

Note: Take care to prevent cross-contamination between template tubes and PCR tubes. Opening template tubes with the left hand, and manipulating PCR lids with the right hand is good practice in preventing sample cross-contamination.

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Final M. musicola M. musicola Internal Reagent M. fijiensis M. eumusae Amplification Concentration (Clade 1) (Clade 6) Control Sterile Water - 8.4µL 8.4µL 8.4µL 6.2µL 8.3µL Primer MM14F (10uM) 300nM 0.6µL 0.6µL - - - Primer MF14F (10uM) 300nM - - 0.6µL - - Primer MYCO200R (10uM) 300nM 0.6µL 0.6µL 0.6µL - - Primer ELS_GEN2_F1 (10uM) 900nM - - - 1.8µL - Primer ELS_GEN2_R1 (10uM) 900nM - - - 1.8µL - Primer MWPL1-110 (10uM) 300nM - - - - 0.6µL Primer MWPL1-197 (10uM) 300nM - - - - 0.6µL Probe MMVIC (10uM) 200nM 0.4µL - - - - Probe MM2 (10uM) 200nM - 0.4µL - - - Probe MF-FAM (10uM) 200nM - - 0.4µL - - Probe ELS_GEN2_as (10uM) 100nM - - - 0.2µL - Probe MWPL1-ROX (10uM) 250nM - - - - 0.5µL RealMasterMix® Probe 2.5X 1X 8µL 8µL 8µL 8µL 8µL Volume pre-template addition 18µL 18µL 18µL 18µL 18µL 18µL

Table 4.3 Mastermix set-up table for Mycosphaerella spp and internal amplification control real-time PCR assays

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REAL‐TIME PCR THERMAL CYCLING PROTOCOL

Reactions may be run immediately or stored at 4ºC until ready. If using a machine other than the RotorGene RG6000, the tubes/plates must be centrifuged prior to cycling. This is particularly important following 4ºC storage as condensation will form in the lids of the tubes.

The thermal cycling regime for the Mycosphaerella spp on banana assays is as follows:

Step Temperature Time Description Other Information (min:sec) 1 94ºC 2:00 Enzyme Activation 2 94ºC 0:15 Denaturation 3 65ºC 0:30 Annealing Acquiring # (59 ºC for ELS) 4 68ºC 0:30 Extension 5 Go to step 2, 44 more times

# Set protocol to acquire during anneal step only.

Using the Corbett Research RG6000 software, channels for data acquisition are:

Target Probe Name Dye Channel M. musicola Clade 1 MMVIC VIC Yellow M. musicola Clade 6 MM2 FAM Green M. fijiensis MF-FAM FAM Green M. eumusae ELS_GEN2_as FAM Green Internal Amplification Control MWPL1-ROX ROX Orange

Note: Channels differ markedly between real-time PCR machines. It is important to check that the appropriate channels are being used for data acquisition. Consult the software manual for this information.

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4.5 RESULTS INTERPRETATION

CONTROL ASSAYS

For the diagnostic assay to be reliable, a number of PCR controls must be included to provide quality assurance against false negative and false positive results.

No Template Controls (NTC) No Template Control or negative control reactions (usually three) must show nil increase in fluorescence above the baseline. The baseline is defined by the level of fluorescence during the initial cycles of PCR. Any appreciable increase in fluorescence by the negative controls is most likely due to template contamination and could result in false positive results. In the event of contamination, the assays should be repeated with fresh aliquots of reagents.

Positive Target Control A positive reaction from the accumulation of target PCR product is indicated by an increase in fluorescence above the baseline. Positive control template must be from a reliable, proven source, in this case DNA extracted from pure fungal culture.

Usually, a positive control reacts strongly (in the CT range 20-30, with a typical amplification plot and with high fluorescence). Failure of the positive target control usually indicates failure of one of the PCR components (enzyme mastermix, primers, probe) and/or may be a result of degradation of the DNA template during storage.

Internal Amplification Control (IAC) This internal amplification control is included to guard against false negatives arising from the DNA extraction technique (commonly caused by presence of PCR inhibitors but also may be due to poor template recovery). An IAC should be set up in parallel for every sample being assayed. Failure of the IAC to amplify renders negative results in the test assays as unreliable.

Note: If the aforementioned controls are all found to be working adequately, proceed to analyse the target concentration standards and unknowns.

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MYCOSPHAERELLA SPP ON BANANA PROBE ASSAYS

The Mycosphaerella assays are highly target specific, and sensitive to at least 50fg of template DNA. Overall fluorescence varies for the reporter dye being used and is commonly lower for probes labelled with VIC (between 0.5-0.6 fluorescence units) than those labelled with FAM (0.8 – 1.0 fluorescence units).

Figure 4.3 illustrates an amplification plot for a standard curve of black Sigatoka. Note the red ‘threshold’ line which is an arbitrary value set by the machine software to provide the best fit of the standard curve through the concentration standards. The threshold line runs through the exponential phase, significantly above the background phase to avoid noise, and below the signal plateau phase. The threshold defines the threshold cycle value (CT), which is the fractional cycle number at the point where the amplification curve crosses a threshold of detection. The intersection of the threshold line and the sample curves provides the CT value for the sample. Plotting the log of the initial target copy number for a set of concentration standards against their CT, results in a straight line. The starting template in unknown samples can then be determined by measuring their CT, and reading off the standard curve (Figure 4.4).

Figure 4.3 Amplification plot of the black Sigatoka standard curve (5ng to 5fg)

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Figure 4.4 Standard curve for determination of starting template number in unknown samples. The graph was generated by plotting the CT against initial target copy number of the standards shown in Figure 4.3

A number of parameters must also be checked when considering the efficiency and relevance of the standard curve data. The correlation coefficient (R2 value) is the percentage of data which matches the hypothesis that the standards form a standard curve. Aim for an R2 value of 0.99. The slope of the reaction (M) can be used to determine the exponential amplification value (10(-1/-3.322)) and the reaction efficiency [10(-1/-3.322)]-1. A 100% efficient reaction occurs when amplification product is doubled in each cycle resulting in an M value of -3.322, an exponential amplification value of 2, and a reaction efficiency of 1 (or 100%).

CALLING A SAMPLE RESULT

When dealing with exotic pathogens, any amplification signal must be considered as a potential positive result. Characteristic amplification plots (showing defined background, exponential and plateau phases) are deemed positive and if all the quality controls are intact, no further analysis is necessary. Amplification plots demonstrating slow kinetics (defined by a long sloping graph and/or no clear plateau phase) may be a result of PCR inhibition or binding of the probe to a cross-specific template. These samples need to be repeated using higher and lower concentrations of template in the reaction, to determine if the effect is due to PCR inhibition. Failure to rectify the problem is likely to indicate a closely related but as yet unidentified Mycosphaerella species and further sequence investigation is warranted. Figure 4.5 illustrates characteristic amplification plots for yellow Sigatoka (definite positives), as

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well as a closely related Mycosphaerella species which is cross-specific for the yellow Sigatoka probe.

Figure 4.5 Amplification plot of yellow Sigatoka positive control (red), definite positive leaf sample (black) and closely related Mycosphaerella species demonstrating slow kinetics (teal). The slow kinetics indicates likely cross-specificity for the yellow Sigatoka probe.

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ACKNOWLEDGEMENT

The editors would like to thank Drs Marie-Françoise Zapater, Françoise Carreel and Jean Carlier for sharing their knowledge and expertise in the morphological diagnosis of banana Mycosphaerella spp. during the 2008 scientific exchange.

REFERENCES

Arzanlou, M, Groenewald, J Z, Fullerton, R A, Abeln, ECA, Carlier, J, Zapater, MF, Buddenhagen, IW, Viljoen, A and Crous, PW (2008). Multiple gene genealogies and phenotypic characters differentiate several novel species of Mycosphaerella and related anamorphs on banana. Persoonia 20,19-37.

Brun J (1963) Cercosporiosis of Banana in Guinea. A study of the ascospore state of M. musicola. Thes. Fac. Sci. Univ. Paris 35, pp 196.

Carlier J, Zapater MF, Lapeyre F, Jones DR, Mourichon X (2000) Septoria leaf spot of banana: A newly discovered disease caused (anamorph Septoria eumusae) by Mycosphaerella eumusae. Phytopathology 90, 884-890.

Crous PW, Mourichon X (2002) Mycosphaerella eumusae and its anamorph Pseudocercospora eumusae spp. nov.: Causal agent of eumusae leaf spot disease of banana. Sydowia 54, 35-43.

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