Effect of Chitosan on Morphological Change Of Colletotrichum capsici (Sydo.
Butler & Bisby Dr.
Ir.
Paristiyanti Nurwardani.
INTRODUCTION
Background Worldwide, postharvest losses have been estimated at 50% and much of this is due to fungal and bacterial infection.
One of the important funguses that attack the postharvest product is fungus Colletotrichum capsici (Sydo.
Butler & Bisby.
This fungus caused anthracnose disease (Figure 1 and .
The symptom of anthracnose is black lesion, usually sunken caused by imperfect fungi that produce conidia in ecervuli.
Conidia are borne on acervuli, witch are erumpent, cushion-like masses of The conidia are hyaline, one celled, avoid to The mycelium of pathogen is septate , inter-and Acervuli and stroma in the stem are hemispherical and 70-120 in diameter.
Setae are scattered and dark brown.
The tips are light brown and several septate and up to 150 in length.
Conidiophores are aseptate and Conidia in mass appear light pinkish in color.
Conidia are borne singly at the tips of the conidiophores.
Individually they are hyaline, unicellular and caved with narrowed ends.
These measure 7-28 x 3-4 .
This is the characteristic oil globule in the center of each conidium.
Conidia germinate in water within four The germ tube soon forms an appressorium (Mehrorta, 1.
Figure 1.
The simptom of anthracnose disease on chili.
Figure 2.
Structure of C.
capsici from chili fruit.
* Dosen Fakultas Pertanian UNSUR Effect of Chitosan on Morphological Change Of Colletotrichum capsici (Sydo.
Butler & Bisby.
Dr.
Ir.
Paristiyanti Nurwardani.
However, growing concerns over the presence of chemical residues in the food chain, the development of fungicide-resistant strain of postharvest pathogen, and the revocation of registration of some of the more effective fungicides, have generated an interest in the development of safer alternatives to synthetic fungicide that are both effective and economically feasible (El-Ghaouth, 1.
Control of postharvest decay was also reported with chitosan.
Chitosan, polysaccharide, is a natural material that appears to play a dual role by interfering directly with fungal growth and by activating several biological processes in plant Chitosan known as antifungal for several fungi, elicitor of many plant defense enzymes, inhibitor for proteinase, and synthesizes of lignin.
According to Benhamou .
, chitosan was found to inhibit radial growth of Fusarium oxysorum radicis-lycopersici with an optimal effect at concentrations ranging from 3 to 6 mg/ml.
Light microscope observations showed morphological changes, including hyphal swelling and distortion.
Objectives radial growth of C.
n-vitro MATERIALS AND METHODS Chitosan preparation:
This research used the pure chitosan .
repared in VEDCALab.
Sheets of chitosan were dissolved in 2.
5% acetic acid and adjusting the pH to 5.
6 with 2 N KOH Five percent .
%) solution of chitosan was solved in 5% acetic acid solution (Figure .
Fungal cultural and growth Pure inoculums of C.
capsici was purification from chili fruit infected by C.
capsici and mantained on Potato Dextrose Agar (PDA) and incubated in 370C incobator in dark place (Figure .
Figure 3.
Resource of chitosan was from shrimp shell The objectives of this research were to determine the direct effect of chitosan on hyphae radial period and interaction between chitosan concentration and hyphae Journal Of Agroscience.
Vol.
1 Th.
1 Juli Ae Desember 2008 Figure 4.
Pure inoculum of C.
Effect of chitosan on colony diameter growth .
ntifungal Potato Dextrose Agar (PDA) was prepared with 0.
025%, 0.
3% ,
75% and 1.
75% chitosan concentration and control.
Every PDA plates was seeded with 5mm-diameter mycelia plug taken from 9-days-old C.
capsici culture.
The treatments were repeated three time.
Inoculated plates were incubated at room temperature in the dark, and fungal growth was recorded at 1-day intervals until the control (PDA without chitosa.
reached the edge of the Growth inhibition is expressed as the percentage of inhibition of radial growth relative to the control.
Effect of chitosan on the morphological change of C.
Colletotrichum capsici did not produce conidia in PDA Plate.
Conidia were produced in Potato Dextrose Chili (PDC-.
liquid Culture of 9-days-old C.
was seeded in 10 ml PDC.
Inoculated PDC flask were cover with aluminum foil and incubated in shaker-water-bath 370C .
Conidia suspension .
l from A 106/m.
were deposited on the surface of microscope slides covered with a thin layer of PDA Chitosan 0.
30, 0.
75, 1.
75% and control (PDA).
Inoculated slide were kept in a moisture chamber .
terile chamber with wet tissue layer Two days after inoculation, all slides were examined by light microscope to asses morphological changes of Effect of chitosan on conidia Conidia suspension .
ml from A 106/m.
was poured into 4 ml PDC-0.
5 liquid medium .
and PDC-0.
5-Chitosan-0.
Every treatment was dropped on specific object glass and the conidia germination was recorded by CCTV microscope.
RESULTS
Effect of chitosan on colony diameter growth .
ntifungal Chitosan significantly (P<0.
reduced the colony diameter growth 9 cm 75% (Table 1.
Figure .
The relative growth 90 Ae 56.
The colony of pathogen at chitosan concentration 0-0.
30% developed actively until by day 9 but at chitosan concentration 0.
were halted.
This research Effect of Chitosan on Morphological Change Of Colletotrichum capsici (Sydo.
Butler & Bisby.
Dr.
Ir.
Paristiyanti Nurwardani.
continued to growth pathogen colony until 14 days, the colony diameter were not growth.
Effect of chitosan on the morphological change of C.
Table 1.
Effect of Chitosan on Colony Diamater Growth and Relative Growth inhibition Microscopic observation show the a coagulation in the fungus cytoplasm characterized by the appearance of small vesicles in hyphae treated by chitosan 0.
75% (Figure 7 a,b,.
At the control treatment (PDA) grew normaly but at all chitosan concentration, hyphae grew in abnormal shapes.
Chitosan 0.
75% induced increase in hyphal swelling, cell lysis and hyphal distortion.
Treatments Colony Diameter .
Relative Growth Inhibition (%) Control Chitosan Chitosan Chitosan Chitosan Means marked with the same letter do not differ significantly (P<0.
Figure 5 : Colony diameter of c.
capsici on chitosan treatment in 9 days after C-0 : control.
C-1: chitosan C-2: chitosan 0.
C-3:
75%, and C-4: chitosan 1.
The linier regression between chitosan concentration and C.
capsici colony diameter was Y= 74 x with R2 = 96.
(P<0.
Every one percent of chitosan in PDA decreased 2.
cm colony diameter (Figure .
Journal Of Agroscience.
Vol.
1 Th.
1 Juli Ae Desember 2008 Colony Diamater = 8.
93- 2.
S = 0.
R-Sq = 97.
R-Sq.
= 96.
Colony Diamater .
Chitosan concentration Figure 6.
Linier regression between chitosan concentration and pathogen colony diameter .
Figure 7.
Morphological change of C.
capsici at different chitosan concentration.
Normal hyphae on PDA had a compact cell wall .
Distortion hyphae on PDA-Chitosan 0.
75% and PDA-Chitosan 1.
75% had swollen hyphae, lyses and disorganization hyphae.
Effect of Chitosan on Morphological Change Of Colletotrichum capsici (Sydo.
Butler & Bisby.
Dr.
Ir.
Paristiyanti Nurwardani.
Conidia germination period In control treatment (PDC-0.
conidia germinated at 9 hours after In the chitosan (PDC-0.
5-Chitosan- 75%) conidia germinated at 68 hours after inoculation and germ tube develops an appressorium or appressorium (Figure .
Figure 8.
Germination process of C.
Conidia germination process in PDC-0,5 .
was 9 hours.
Conidia germination process in PDC-0,5-Chitosan 0,75% was 68 hours.
Journal Of Agroscience.
Vol.
1 Th.
1 Juli Ae Desember 2008 DISCUSSION