Molecular Monitoring of Wild-Type and Genetically Engineered Colletotrichum coccodes Biocontrol Strains In Planta

Area of Science:

• Plant Pathology
• Molecular Biology
• Mycology

Background:

• Colletotrichum coccodes is a fungal pathogen affecting various plants.
• Genetic engineering aims to enhance fungal virulence for biological control applications.
• Velvetleaf (Abutilon theophrasti) is a significant agricultural weed.

Purpose of the Study:

• To monitor the in planta colonization of wild-type and NEP1-engineered Colletotrichum coccodes strains in velvetleaf.
• To quantitatively assess fungal growth and plant response using molecular techniques.
• To investigate the expression of the necrosis- and ethylene-inducing peptide (NEP1) transgene.

Main Methods:

• Real-time quantitative polymerase chain reaction (QPCR) with SYBR Green chemistry was used for fungal DNA quantification.
• Detection of host plant DNA was employed to monitor plant tissue degradation.
• Conventional retro-transcription polymerase chain reaction (RT-PCR) was used to assess NEP1 transgene expression.

Main Results:

• QPCR successfully quantified both C. coccodes strains from the time of inoculation up to 14 days post-infection.
• Increased fungal DNA correlated with the development of necrotic lesions caused by the wild-type strain.
• The wild-type C. coccodes demonstrated greater infectivity on velvetleaf compared to the T-20a strain.
• NEP1 transgene expression was not detected in the T-20a strain, suggesting gene silencing.
• Decreased plant DNA levels were observed in response to wild-type C. coccodes infection.

Conclusions:

• QPCR is a reliable method for in planta monitoring of fungal pathogens, detecting them before symptom appearance.
• The wild-type C. coccodes is more virulent on velvetleaf than the NEP1-engineered strain.
• Silencing of the NEP1 transgene may explain the reduced virulence of the engineered strain.