Enhanced resistance to Phoma tracheiphila and Botrytis cinerea in transgenic lemon plants expressing a Trichoderma harzianum chitinase gene

Abstract

Antifungal genes from micro-organisms were inserted into the genome of a number of plant species, representing a promising strategy for conferring genetic disease resistance against a broad range of plant pathogenic fungi. In the present study, the chit42 gene from Trichoderma harzianum (codifying the antifungal protein endochitinase) was introduced into the ÔFemminello siracusanoÕ lemon by Agrobacterium tumefaciens, in order to regenerate transgenic plants resistant to fungal disease. Three polymerase chain reaction (PCR)-positive clones were obtained. Southern blot confirmed the integration of the transgene in the lemon genome and revealed that one or two copies had been inserted. Reverse transcriptase-PCR, Northern blot and Western analysis were performed and the results confirmed the expression of the inserted gene. The transgenic clones were tested in vitro and in vivo for disease resistance. Conidia germination and fungal growth of Phoma tracheiphila were strongly inhibited in vitro by the transgenic foliar proteins, while no effects were observed with the controls. Disease resistance assays were performed in vivo with Botrytis cinerea, the causal agent of grey mould in fruit. Transgenic lemon plants, inoculated with lemon petals infected by a single-conidial isolate of B. cinerea, showed significantly less lesion development than the controls. On the whole, the results indicate that the transformation with the antifungal endochitinase gene may represent a strategy for disease control in citrus crops.

Plant-beneficial effects of Trichoderma and of its genes

“…chit42 expression increased resistance to Venturia inaequalis, but reduced plant growth, in apple (Bolar et al, 2000) and significantly increased the resistance of broccoli to attack by Alternaria brassicicola (Mora & Earle, 2001). The expression of chit42 in lemon enhanced resistance to Phoma tracheiphila and B. cinerea, a significant correlation between resistance and transgene expression being observed, with an upregulation of ROS and JA/ET-responsive genes (Gentile et al, 2007;Distefano et al, 2008). The homologous chit42 gene from T. virens was able to enhance resistance against R. solani when it was expressed in rice (Shah et al, 2009).…”

In vitro screening of guava plantlets transformed with endochitinase gene against Fusarium oxysporum f.sp. psidii

“…Development of a transgenic guava variety with genes such as endochitinase which encodes enzymes which can degrade the fungal cell wall could prove to be a permanent solution to this problem. The technique of expressing the endochitinase gene in the plant system to confer resistance against fungal diseases has been suc-doi: 10.17221/74/2015-CJGPB cessfully demonstrated in apple (Bolar et al 2000), tomato (Girhepuje & Shinde 2011), cotton (Emami et al 2003;Cheng et al 2005), broccoli (Mora & Earle 2001), lemon (Gentile et al 2007) and rice (Lu et al 2004;Shah et al 2009). Chitinases have been shown to possess an antifungal role in disease resistance (Broglie et al 1991;Sela-Buurlage et al 1993).…”

Transformation of canola by chit33 gene towards improving resistance to Sclerotinia sclerotiorum

“…The chitinase enzymes are fungicidal (Jayaraj & Punja 2007;Xiao et al 2007) and non-toxic to plants, animals, and higher vertebrates (Lorito et al 1996). Some research has been conducted to develop transgenic crop plants that have increased expression levels of chitinase genes in hopes of producing fungal disease resistant varieties (Liu et al 2004;Deng et al 2007;Gentile et al 2007;Jayaraj & Punja 2007;Xiao et al 2007). It has been shown that chitinase enhances fungal disease resistance in different species (Gentile et al 2007), and rice against Rhizoctonia solani and Magnaporthe grisea (Liu et al 2004).…”

“…Some research has been conducted to develop transgenic crop plants that have increased expression levels of chitinase genes in hopes of producing fungal disease resistant varieties (Liu et al 2004;Deng et al 2007;Gentile et al 2007;Jayaraj & Punja 2007;Xiao et al 2007). It has been shown that chitinase enhances fungal disease resistance in different species (Gentile et al 2007), and rice against Rhizoctonia solani and Magnaporthe grisea (Liu et al 2004). Also tobacco transformants overexpressing chit33 showed a noticeable tolerance to biotic stress like fungal and bacterial pathogens as well as abiotic stress such as high salinity and heavy metal contamination (Dana et al 2006).…”

“…Theoretically, the foreign genes can be transferred into the genomes of plants without altering any other agro-economically important traits (Chang et al 2002). Since chitin is a structural polymer in many fungal pathogens, the chitinases which are potential antifungal agents through their chitin degradation activity, are excellent candidates for controlling the fungal pathogens development (Kalai et al 2006;Maximova et al 2006;Gentile et al 2007;Jayaraj & Punja 2007;Xiao et al 2007;Pasonen et al 2008).…”