Protocol to produce and quantify conidial anastomosis tubes (CAT) fusion in Colletotrichum lindemuthianum

Francine H. Ishikawa and Gabriela Roca

francine.hiromi@univasf.edu.br

G.Roca@tu-braunschweig.de

Background

Conidial anastomosis tubes (CATs) are specialized cell-type that allows fusion between conidia. Colletotrichum lindemuthianum naturally undergoes CAT fusion on the bean plant leaf surface (Ishikawa et al., 2010a) and within asexual fruiting bodies in anthracnose lesions on its host (Roca et al., 2003). We have being using the following protocol to produce and analyse CAT fusion in vitro with robust results (Ishikawa et al., 2010b) (Fig 1). CAT fusion in this pathogen was found to exhibit significant differences to the model system Neurospora crassa (Ishikawa et al., 2010b). In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer, conditions that are not commonly used experimentally.

Fig 1. CAT fusion of LV115 strain of C. lindemuthianum in water.  The conidia were harvested from 16 day old bean pod agar cultures. (A) After 24 hrs of incubation (B) After 72 hrs. Arrows indicated fusions between conidia/conidial germlings via CATs. Note the appressorium formation (asterisk). Bar: 10µm.

Protocol

1)           Grow Colletotrichum lindemuthianum for sporulation. We use glass tubes containing autoclaved French bean pods submerged in 2% water agar (bean pod agar medium). Incubated in the dark at 22ºC.

2)           Collect conidia from 10-16 day old tube cultures. CAT fusion is strain dependant, so we suggest to use 15 days old spores to start and the culture age could be adapted depending of the results obtained. Our previous results showed that older conidia fuse more frequently and quicker than younger conidia, however, timing of CAT fusion is strain dependent – (see Ishikawa et al. 2010b for more). Suspend them in water. First fill an Ependorf tube with 1 ml of sterilized water, then collect the conidia (pink mass) and mix in the water. Note the time. Your measurement of CAT fusion will be 24 hours after this point.

3)           Rapidly shake the Eppendorf tubes on vortex for a few seconds to produce a suspension of dissociated conidia.

4)           Measure the concentration of conidia in your suspension.

5)           Diluted your suspension to a concentration of 2 x 106 conidia/ml (conidial work suspension). Ensure that you have at least 600 µl of suspension.

6)           Place 200 µl of conidial suspension into 3 separate compartments of a slide culture chamber (we use an eight-well slide culture chamber from Nalge Nunc International, Rochester, NY). You will therefore have 3 x 200 µl samples to analyse.

7)           Place the culture chamber in an incubator in the dark at 22ºC until the first measurement to be made after 24 h incubation. C. lindemuthianum germination and CAT formation is slow, thus the next measurement point could be after 12-24 h. If the objective is to follow all process of CAT fusion, you may need to start between 24-48 h of incubation . Higher number of fusions (chain of fused conidia) is observed after 72 h of incubation (Fig. 1 B).

8)           Examine the samples at room temperature by using brightfield or differential interference contrast optics with a 60x objective . We need an inverted microscope because our cover glass slide are on the bottom. If you have and upright microscope you would need to adapt the slide sample preparation

9)           Quantify CAT fusion as the percentage of conidia or conidial germlings involved in fusion. For each strain and time point, use 3 samples (the 3 chambers you filled with conidial suspension) and quantify 100 conidia in each sample (n=300). You can record 10-15 images per sample condition for subsequent analysis. Repeat these three times (i.e. another days but use always the same conidial age). Estimate mean and standard deviation (SD).

10)        Incubate the samples at 22°C between measurements. Be very careful not to shake samples when transferring them between the incubator and microscope as this may perturb CAT fusion.

11)        Produce a graph of conidial germination and CAT fusion at different time points.

References

Ishikawa F.H., Barcelos Q.L., Alves E.A., Camargo Jr. O.A., Souza E.A., 2010a. Symptoms and pre-penetration events associated with the infection of common bean by the anamorph and teleomorph of Glomerella cingulata f. sp. phaseoli. Journal of Phytopathology 158: 270-277.

Ishikawa F. H., Souza E. A., Read N. D, Roca M..G., 2010b. Live-cell imaging of conidial fusion in the bean pathogen Colletotrichum lindemuthianum. Fungal Biology, 114: 2-9.

Roca M.G., Davide L.C., Mendes-Costa M.C., Wheals A., 2003. Conidial anastomosis tubes in Colletotrichum. Fungal Genetics and Biology 40:138-145.

Colletotrichum Workshop II 2010: Colletotrichum: Species, ecology and interactions

As a response to the rapidly increasing interest in Colletotrichum systematics, pathology and genetics, the SECOND 2010 Colletotrichum workshop will take place on 1 August at the Edinburgh International Conference Centre, as a satellite meeting to IMC9, organized by Paul Cannon (p.cannon@cabi.org) and Ulrike Damm (u.damm@cbs.knaw.nl). Participants will need to register at the conference website (http://www.imc9.info) but do not have to pay for the entire meeting. Contact Paul or Ulrike, especially if you would like to make a short presentation about your work.

Species concepts in the economically important genus Colletotrichum have been undergoing revolutionary change in recent years. The old morphology-based species are rapidly being superseded by taxa that are largely defined by molecular sequence. Cryptic species are increasingly being recognized that cannot be reliably separated using morphological or cultural methods. In a number of cases, however, these demonstrably monophyletic units do not show marked host preference or shared pathogenic characteristics. This apparent paradox reduces the utility of the new classifications to the applied communities, especially to pathologists. We also know too little about the evolutionary significance of the varied nutritional/pathological strategies shown by Colletotrichum, especially the mechanisms by which colonies change status from endophyte/hemibiotroph to aggressive pathogen on the one hand, or to saprotroph on the other.

There will also be opportunities to exchange information relating to the rapidly increasing number of whole-genome sequencing projects that are planned or already in operation.

The meeting will bring together key workers from the academic and applied sectors to share knowledge, review progress and set priorities for further studies.

Colletotrichum Workshop I 2010

Title: Colletotrichum Workshop 2010

Organizers:

Michael Thon, University of Salamanca

Serenella Sukno, University of Salamanca

Location:

The meeting will take place as part of the 10th European Conference on Fungal Genetics  (ECFG10)  at NH Conference Centre Leeuwenhorst, Netherlands.   For more information about the conference, please see this page: at http://www.ecfg10.info/index.php

Data and Time: Monday 29 of March, 1:00 – 5:00 PM

Program:

1 PM opening words Dr. Michael Thon

1.10 PM Dr. Yasuyuki Kubo

Novel peroxin, Fam1p of Colletotrichum orbiculare is essential for pathogenesis and associates with Woronin bodies

1. 35 PM  Jochen Kleemann

Identification and characterization of candidate effector proteins secreted by the crucifer anthracnose fungus Colletotrichum higginsianum

2 PM Dr. Elaine Souza

Genetics analysis in Colletotrichum lindemuthianum

2.25 PM Riccardo Baroncelli

Evolutionary relationships in Colletotrichum acutatum populations

2.45.PM Break 30 minutes.

3.15 PM Emiel Ver Loren van Themaat

Towards a high quality genome and transcriptome of Colletotrichum higginsianum

3.40 PM Dr. Li Ju Ma/ Dr. L. Vaillancourt

Status of the C. graminicola genome sequence

4.05 PM Dr. Michael R. Thon

Genome wide computational analysis of C. graminicola for secreted proteins

4.30 PM discussion of genome annotation

Colletotrichum Workshop 2009

We are finalizing our plans to have a Colletotrichum workshop during the fungal genetics conference at Asilomar.  Here are the details:
Title: Colletotrichum Workshop
Organizers:
Michael Thon, University of Salamanca
Serenella Sukno, University of Salamanca
Location:
The meeting will take place as part of the Twenty Fifth Fungal Genetics Conference, Asilomar Conference Grounds, Pacific Grove, California, USA. For more information about the conference, please see this page:
Meeting Romm: Scripps Building on the Asilomar Conference Grounds.
Data and Time: Wednesday, March 18th, 12:15 – 2:00 PM
Program:

12:15-12:20 Introduction

12:20-12:40 Lijun Ma – Status of the C. graminicola genome sequencing project.

12:40-1:00 Stephen Wirsel – Colletotrichum graminicola causes contrasting symptoms and differentially affects photosynthesis, senescence and host gene expression when infecting mature or senescing corn leaves.

1:00-1:20 Jo Ann Crouch – Will genomics solve the riddle of Colletotrichum sex determination?

1:20-1:40 Michael Thon – The Fungal Gene Cluster Database and other topics

1:40-2:00 Discussion – C. graminicola genome annotation and other topics.

Box lunches will be provided for workshop attendees.  Note: There will be 20 box lunches available for pre-registered participants.
Registration:
If you would like to attend, please send me an email and let me know.  Even if you have already replied to my previous email, please send me an email again to confirm that you can attend.  We need to have an accurate count of attendees so we can order a large enough room and have enough box lunches.
Thanks!
Mike and Serenella