Priors and Justifications

Bean is one of Brazils most important annual crops, both social and
economically. Among the factors affecting its sustainability we can highlight plague and
disease occurrence. Moreover, the fungal diseases are the ones which cause the worst
troubles.

Anthracnose and scab are both caused by species from the genus

Colletotrichum, in the anamorphic form, and Glomerella, in the teleomorphic form.

Bean anthracnose holds a wide variability described between races and in
the same race (PINTO et al., 2012). Recent researches report strains of
C.lndemunthianum and Glomerella sp. obtained from lesions caused by bean anthracnose
(BARCELOS et al., 2014). Comparative analysis of morphological and molecular
characters and pathogenicity of those strains have shown high variability and pointed that
Glomerella sp. strains do not represent the teleomorphic form of C.lindemuthianum
(BARCELOS et al., 2014). Those authors identified two distinguished grous of
Glomerella sp. strains collected from bean-anthracnose lesions in bean, called Glomerella
sp. groups I and II. Group Glomerella sp. amplifies for the primers HMGglo
(BARCELOS et al., 2011), doesnt amplify for the pair of specific C.lindemuthianum
primers (GARCIA-SERRANO et al., 2008) and dont cause any symptoms in bean.
However a small group of six strains on group II doesnt amplify for both primers but
causes mild symptoms on the bean. Mota, 2013, using those strains from group II, and
other strains from bean-scab lesions, noticed that both presented similar morphological
and cytological characteristics and cause bean-scab symptoms. Moreover, those strains
form conidial anastomosis tubes (CATs). Cat formation is considered a potential
mechanism for sexual recombination and horizontal gene transfer. The occurrence of
strains which cause bean-anthracnose and scab in the same lesion on the bean and which
belong to the genus Colletotrichum may favor CAT formation between those strains; it is
important from the evolutionary point of view and also to understand the interaction of
those pathogens with the bean. There is shortage of information about bean scab, because
it is an emerging disease on bean.
Thus, the evaluation of the nuclear dynamics during the CAT fusion process
between and in the strains of Colletothrichum (Glomerella) affecting beans and also of
their karyotypes become important to determine if those species have any genetic
material in common, acquiring biological information on those pathogens. Moreover, that

information will help understand the emergence of new diseases on bean and the control
strategies for them, specially through genetic resistance.

Objetivos

To analyze nuclear dynamics during CAT fusion process between the species

belonging to the genus Colleothrichum;

To study cellular communication through MAP-kinase (MAK-2) protein on the

species.
To obtain recombines strains between and in the species.
To compare the karyotype of the species from the genus Colletotrichum and the

recombinant strains through PFGE e GTBM;

To evaluate pathogenicity of the parental and recombinant strains.

Materials and Methods

We will use Colleotrichum lindemuthianum strains, causal agent of anthracnose,
Glomerella cingulate f. sp. phaseoli from bean-anthracnose lesions and Colltotrichum
spp isolated, which cause bean scab. Every strain belongs to the Mycology collection of
the Laboratory of Plant Resistance to Diseases, in the Biology department of Federal
University of Lavras.
We will use two plasmids, the first, pGR02, containing the tdimerRed gene, which
express red fluorescent protein (RFP) fused with the Histone H4-2 gene (FDSG_05491.3)
and the gene which grants phleomycin resistance (Phleo). The second plasmid, pMF357,
contain the histone H1 fused with sgfp gene, which expresses green fluorescent protein
(GFP) and the gene which grants resistance to hygromycin (Hyg) (ISHIKAWA et al.,
2012).
The protoplasts will be obtained through modified Ishikawa methodology (2010b)
and the transformations will be carried out using the methodology described by Ishikawa
et al. (2010).

CAT fusion will be evaluated between and in the different species and the nuclear
dynamics, later the recombinant among the different species will be selected according to
the methodology described by Ishikawa et al., 2012.
The study of the cellular communication will be carried out according to the
methodology proposed by Fleissner et al., 2009.
The chromosome number of the parental strains and the recombinant strains will
be evaluated through the method of Pulsed-Field Gel Electrophoresis (PFGE), according
to the modified methodology of O Sullivan et al., 1998, and through Germ tub burst
Method (GTBM), according to the modified methodology of Taga et al., 1998. The
pathogenicity of the parental and recombinant strains will be evaluated according to the
methodology of Ishikawa et al., 2012.
Results
The first step is to obtain the strains, so until now one strain of Colletotrichum
lindemuthianum is transformed with the plasmid pMF357. The remaining strains are
being transformed; however we are finding some difficulties in the plasmid integration to
the fungus genome.
The strain pathogenicity has been determined, some cause symptoms to the bean,
and others do not.
The species karyotyping through GTBM is being carried out successfully through
PFGE, the methodology is being optimized.