Research Article: In vitro production of two chitinolytic proteins with an inhibiting effect on the insect coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) and the fungus Hemileia vastatrix the most limiting pests of coffee crops

Date Published: March 30, 2012

Publisher: Springer

Author(s): Claudia P Martínez, Claudia Echeverri, Juan C Florez, Alvaro L Gaitan, Carmenza E Góngora.


Two genes from Streptomyces albidoflavus, one exochitinase (905-bp) and an endochitinase (1100-bp) were functionally expressed in Escherichia coli in form of a fusion protein with a maltose binding protein (MBP). The goal was to produce and test proteins that inhibit both the coffee berry borer insect Hypothenemus hampei and the coffee rust fungus Hemileia vastatrix. Both recombinant proteins MBP/exochitinase and MBP/endochitinase showed chitinolytic activity. When recombinant purified proteins were added to an artificial coffee-based diet for the coffee berry borer, MBP/exochitinase at a concentration of 0.5% W/W caused delayed growth of larvae and 100% mortality between days 8 and 15, while MBP/endochitinase caused 100% mortality at day 35. H. vastatrix urediniospores presented total cell wall degradation in their germinative tubes within 18 h of exposure to the proteins at enzyme concentrations of 5 and 6 mg ml-1, with exochitinase having the greatest effect. The dual deleterious effect of S. albidoflavus chitinases on two of the most limiting coffee pests worldwide, the coffee borer and the coffee rust, make them potential elements to be incorporated in integrated control strategies.

Partial Text

Colombia is one of the most important countries for production of mild coffee (Coffea arabica L.), with over 870,000 Ha planted (Federacafé 2010). The coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytidae), is the most significant pest of the Colombian coffee crop, and it is found in all the coffee-growing regions of the world (Benavides et al. 2012; Bustillo 2002). Female insects fly towards coffee beans and bore into them until reaching the seed endocarpium, where they deposit their eggs. After the eggs hatch, larvae feed on the seed, causing weight loss in the grain, decreasing quality due to fungal contamination, and the falling of small cherries to the ground (Duque et al. 1997).

Based on the DNA and predicted protein sequences analysis of both enzymes, 384 aa (out of 655) of the endochitinase protein showed 95% identity and E value = 0 to a segment of the C terminal region of Chitinase C from Streptomyces albus J1074 (Accession number D6B6V8, Uniprot). Meanwhile, 320 aa (out of 345) of the exochitinase exhibited 72% identity with E value of 1.0 × 10 -117 to a portion of a putative Chitinase A (Accession number F3ZBD7, Uniprot) from Streptomyces sp. Tu6071.

Historically, control of insect pests and diseases in commercial crops has been predominantly based on the use of pesticides. Current environmental trends and developments restrain the use of synthetic chemicals and procure the application of alternatives that complement an integrated pest management approach that also includes genetic, biological and cultural control measures. This is especially true in semi-perennial agricultural systems like coffee, where it is common to find plantations over 20 years old, closely associated to a diverse combination of plant, animal and microbial species. The identification of proteins that could be introduced in semi-perennial crops to generate insect and pathogen resistant plants is important for the sustainability of the agronomical production. Because of their target, chitinases are suitable candidates among those proteins.

The authors declare that they have no competing interests.




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