Applied and Environmental Microbiology, Jul 17, 2023
Pesticidal proteins derived from bacteria such as Bacillus thuringiensis are valuable tools for m... more Pesticidal proteins derived from bacteria such as Bacillus thuringiensis are valuable tools for management of agricultural insect pests and provide a sustainable alternative to the application of chemical insecticides. However, relatively few bacterial pesticidal proteins have been used for suppression of hemipteran or sap-sucking insects such as the Asian citrus psyllid, Diaphorina citri .
Citrus is one of the top fruit crops worldwide. Citrus production faces many challenges such as d... more Citrus is one of the top fruit crops worldwide. Citrus production faces many challenges such as diseases, insects, and abiotic stresses. Genetic improvement of citrus using conventional breeding is a lengthy, costly, and time-consuming process. Biotechnological approaches such as Agrobacterium-mediated transgenic expression, Citrus tristeza virus (CTV)-mediated transient expression and CRISPR-based genome editing have shown tremendous potential to improve citrus against different diseases. Here, we summarize the progress in generating disease-resistant/tolerant citrus plants via biotechnological approaches.
Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibact... more Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibacter americanus (CLam) and Candidatus Liberibacter asiaticus (CLas), which are phloem-restricted and associated with the most important and destructive worldwide citrus disease, Huanglongbing (HLB). Currently, no cure for HLB has been described. Therefore, measures have focused on reducing D. citri populations. In these insects, cathepsin B (DCcathB) and L (DCcathL) enzymes play an important role in digestion, and are involved in embryogenesis, immune defense, and ecdysis. In this study, we used a CTV-based vector to deliver dsRNA (CTV-dsRNA) into Citrus macrophylla plants targeting DCcathB and DCcathL genes in D. citri that fed on the phloem of these CTV-RNAi infected plants. Subsequently, we evaluated expression of DCcathB and DCcathL genes as well as the Vitellogenin (Vg) gene by RT-qPCR in D. citri fed on CTV-dsRNA occurring in plant phloem. It was found that a defective phenotype in D...
The Asian citrus psyllid, Diaphorina citri, vectors the bacterial causative agent of citrus green... more The Asian citrus psyllid, Diaphorina citri, vectors the bacterial causative agent of citrus greening disease, which has severely impacted citrus production on a global scale. As the current repeated application of chemical insecticides is unsustainable for management of this insect and subsequent protection of groves, we investigated the potential use of the bacteria-derived pesticidal protein, Cry1Ba1, when delivered via transgenic citrus plants. Having demonstrated transformation of the Indian curry leaf tree, Bergera koenigii, for Cry1Ba1 expression for use as a trap plant, we produced transgenic plants of Duncan grapefruit, Citrus paridisi, Valencia sweet orange, Citrus sinensis, and Carrizo citrange, C. sinensis x Poncirus trifoliata, for expression of Cry1Ba1. The presence of the cry1ba1 gene, and cry1ba1 transcription were confirmed. Western blot detection of Cry1Ba1 was confirmed in most cases. When compared to those from wild-type plants, leaf discs from transgenic Duncan a...
Stem pitting is a complex and economically important virus-associated disease of perennial woody ... more Stem pitting is a complex and economically important virus-associated disease of perennial woody plants. Molecular mechanisms and pathways occurring during virus-plant interaction that result in this phenomenon are still obscure. Previous studies indicated that different Citrus tristeza virus (CTV) mutants induce defined stem pitting phenotypes ranging from mild (CTVΔp13) to severe (CTVΔp33) in Citrus macrophylla trees. In this study, we conducted comparative transcriptome analyses of C. macrophylla trees infected with CTV mutants (CTVΔp13 and CTVΔp33) and a full-length virus in comparison to healthy plants as control. The mild CTV stem pitting mutant had very few differentially expressed genes (DEGs) related to plant defense mechanism and plant growth and development. In contrast, substantial gene expression changes were observed in plants infected with the severe mutant and the full-length virus, indicating that both the p13 and p33 proteins of CTV acted as a regulator of symptom ...
Plant viruses are threatening many valuable crops, and Citrus tristeza virus (CTV) is considered ... more Plant viruses are threatening many valuable crops, and Citrus tristeza virus (CTV) is considered one of the most economically important plant viruses. CTV has destroyed millions of citrus trees in many regions of the world. Consequently, understanding of the transmission mechanism of CTV by its main vector, the brown citrus aphid, Aphis (Toxoptera) citricidus (Kirkaldy), may lead to better control strategies for CTV. The objective of this study was to understand the CTV–vector relationship by exploring the influence of viral genetic diversity on virus transmission. We built several infectious clones with different 5′-proximal ends from different CTV strains and assessed their transmission by the brown citrus aphid. Replacement of the 5′- end of the T36 isolate with that of the T30 strain (poorly transmitted) did not increase the transmission rate of T36, whereas replacement with that of the T68-1 isolate (highly transmitted) increased the transmission rate of T36 from 1.5 to 23%. Fi...
Fusarium vascular wilt, caused by Fusarium oxysporum f. sp. lycopersici, affects tomatoes worldwi... more Fusarium vascular wilt, caused by Fusarium oxysporum f. sp. lycopersici, affects tomatoes worldwide. Development of resistant varieties of tomato would constitute an economically and environmentally sound approach for the management of this disease. Resistance genes to F. oxysporum f. sp. lycopersici race 1 (I-1 gene) and race 2 (I-2 gene) were mapped to chromosome 11. The I-2 gene cluster includes one functional copy and six nonfunctional homologs of the I-2 gene. This report describes the design of primers based on the functional gene copy and the development of a multiplex polymerase chain reaction (PCR)-based method that has the ability to differentiate I-2 genotypes from genotypes without the I-2 gene. In these trials, 39 of the 40 genotypes tested with known reactions to race 2 gave the expected results. The only exception was the cultivar Plum Crimson carrying the I-3 gene for resistance, which confers resistance to F. oxysporum f. sp. lycopersici races 1, 2, and 3. This meth...
The curry leaf tree, Bergera koenigii, is highly attractive to the Asian citrus psyllid, Diaphori... more The curry leaf tree, Bergera koenigii, is highly attractive to the Asian citrus psyllid, Diaphorina citri, which vectors the bacterial causative agent of citrus greening or huanglongbing disease. This disease has decimated citrus production in Florida and in other citrus-producing countries. As D. citri exhibits high affinity for feeding on young leaves of B. koenigii, transgenic B. koenigii expressing bacteria-derived pesticidal proteins such as Cry1Ba1 have potential for D. citri management when planted in or adjacent to citrus groves. Importantly, the plant pathogenic bacterium that causes citrus greening does not replicate in B. koenigii. Transgenic plants of B. koenigii were produced by insertion of the gene encoding the active core of the pesticidal protein Cry1Ba1 derived from Bacillus thuringiensis. The transformation success rate was low relative to that of other citrus, at 0.89%. T-DNA integration into the genome and cry1ba1 transcription in transgenic plants were confirme...
Table S1. Identify homology of CHS or CHS-like genes considered in Fig. 1. Table S2. Information ... more Table S1. Identify homology of CHS or CHS-like genes considered in Fig. 1. Table S2. Information of the CHS candidate genes and primers used for expression analysis. Table S3. Standard curve used in flavonoids detection. (DOCX 127 kb)
Applied and Environmental Microbiology, Jul 17, 2023
Pesticidal proteins derived from bacteria such as Bacillus thuringiensis are valuable tools for m... more Pesticidal proteins derived from bacteria such as Bacillus thuringiensis are valuable tools for management of agricultural insect pests and provide a sustainable alternative to the application of chemical insecticides. However, relatively few bacterial pesticidal proteins have been used for suppression of hemipteran or sap-sucking insects such as the Asian citrus psyllid, Diaphorina citri .
Citrus is one of the top fruit crops worldwide. Citrus production faces many challenges such as d... more Citrus is one of the top fruit crops worldwide. Citrus production faces many challenges such as diseases, insects, and abiotic stresses. Genetic improvement of citrus using conventional breeding is a lengthy, costly, and time-consuming process. Biotechnological approaches such as Agrobacterium-mediated transgenic expression, Citrus tristeza virus (CTV)-mediated transient expression and CRISPR-based genome editing have shown tremendous potential to improve citrus against different diseases. Here, we summarize the progress in generating disease-resistant/tolerant citrus plants via biotechnological approaches.
Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibact... more Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibacter americanus (CLam) and Candidatus Liberibacter asiaticus (CLas), which are phloem-restricted and associated with the most important and destructive worldwide citrus disease, Huanglongbing (HLB). Currently, no cure for HLB has been described. Therefore, measures have focused on reducing D. citri populations. In these insects, cathepsin B (DCcathB) and L (DCcathL) enzymes play an important role in digestion, and are involved in embryogenesis, immune defense, and ecdysis. In this study, we used a CTV-based vector to deliver dsRNA (CTV-dsRNA) into Citrus macrophylla plants targeting DCcathB and DCcathL genes in D. citri that fed on the phloem of these CTV-RNAi infected plants. Subsequently, we evaluated expression of DCcathB and DCcathL genes as well as the Vitellogenin (Vg) gene by RT-qPCR in D. citri fed on CTV-dsRNA occurring in plant phloem. It was found that a defective phenotype in D...
The Asian citrus psyllid, Diaphorina citri, vectors the bacterial causative agent of citrus green... more The Asian citrus psyllid, Diaphorina citri, vectors the bacterial causative agent of citrus greening disease, which has severely impacted citrus production on a global scale. As the current repeated application of chemical insecticides is unsustainable for management of this insect and subsequent protection of groves, we investigated the potential use of the bacteria-derived pesticidal protein, Cry1Ba1, when delivered via transgenic citrus plants. Having demonstrated transformation of the Indian curry leaf tree, Bergera koenigii, for Cry1Ba1 expression for use as a trap plant, we produced transgenic plants of Duncan grapefruit, Citrus paridisi, Valencia sweet orange, Citrus sinensis, and Carrizo citrange, C. sinensis x Poncirus trifoliata, for expression of Cry1Ba1. The presence of the cry1ba1 gene, and cry1ba1 transcription were confirmed. Western blot detection of Cry1Ba1 was confirmed in most cases. When compared to those from wild-type plants, leaf discs from transgenic Duncan a...
Stem pitting is a complex and economically important virus-associated disease of perennial woody ... more Stem pitting is a complex and economically important virus-associated disease of perennial woody plants. Molecular mechanisms and pathways occurring during virus-plant interaction that result in this phenomenon are still obscure. Previous studies indicated that different Citrus tristeza virus (CTV) mutants induce defined stem pitting phenotypes ranging from mild (CTVΔp13) to severe (CTVΔp33) in Citrus macrophylla trees. In this study, we conducted comparative transcriptome analyses of C. macrophylla trees infected with CTV mutants (CTVΔp13 and CTVΔp33) and a full-length virus in comparison to healthy plants as control. The mild CTV stem pitting mutant had very few differentially expressed genes (DEGs) related to plant defense mechanism and plant growth and development. In contrast, substantial gene expression changes were observed in plants infected with the severe mutant and the full-length virus, indicating that both the p13 and p33 proteins of CTV acted as a regulator of symptom ...
Plant viruses are threatening many valuable crops, and Citrus tristeza virus (CTV) is considered ... more Plant viruses are threatening many valuable crops, and Citrus tristeza virus (CTV) is considered one of the most economically important plant viruses. CTV has destroyed millions of citrus trees in many regions of the world. Consequently, understanding of the transmission mechanism of CTV by its main vector, the brown citrus aphid, Aphis (Toxoptera) citricidus (Kirkaldy), may lead to better control strategies for CTV. The objective of this study was to understand the CTV–vector relationship by exploring the influence of viral genetic diversity on virus transmission. We built several infectious clones with different 5′-proximal ends from different CTV strains and assessed their transmission by the brown citrus aphid. Replacement of the 5′- end of the T36 isolate with that of the T30 strain (poorly transmitted) did not increase the transmission rate of T36, whereas replacement with that of the T68-1 isolate (highly transmitted) increased the transmission rate of T36 from 1.5 to 23%. Fi...
Fusarium vascular wilt, caused by Fusarium oxysporum f. sp. lycopersici, affects tomatoes worldwi... more Fusarium vascular wilt, caused by Fusarium oxysporum f. sp. lycopersici, affects tomatoes worldwide. Development of resistant varieties of tomato would constitute an economically and environmentally sound approach for the management of this disease. Resistance genes to F. oxysporum f. sp. lycopersici race 1 (I-1 gene) and race 2 (I-2 gene) were mapped to chromosome 11. The I-2 gene cluster includes one functional copy and six nonfunctional homologs of the I-2 gene. This report describes the design of primers based on the functional gene copy and the development of a multiplex polymerase chain reaction (PCR)-based method that has the ability to differentiate I-2 genotypes from genotypes without the I-2 gene. In these trials, 39 of the 40 genotypes tested with known reactions to race 2 gave the expected results. The only exception was the cultivar Plum Crimson carrying the I-3 gene for resistance, which confers resistance to F. oxysporum f. sp. lycopersici races 1, 2, and 3. This meth...
The curry leaf tree, Bergera koenigii, is highly attractive to the Asian citrus psyllid, Diaphori... more The curry leaf tree, Bergera koenigii, is highly attractive to the Asian citrus psyllid, Diaphorina citri, which vectors the bacterial causative agent of citrus greening or huanglongbing disease. This disease has decimated citrus production in Florida and in other citrus-producing countries. As D. citri exhibits high affinity for feeding on young leaves of B. koenigii, transgenic B. koenigii expressing bacteria-derived pesticidal proteins such as Cry1Ba1 have potential for D. citri management when planted in or adjacent to citrus groves. Importantly, the plant pathogenic bacterium that causes citrus greening does not replicate in B. koenigii. Transgenic plants of B. koenigii were produced by insertion of the gene encoding the active core of the pesticidal protein Cry1Ba1 derived from Bacillus thuringiensis. The transformation success rate was low relative to that of other citrus, at 0.89%. T-DNA integration into the genome and cry1ba1 transcription in transgenic plants were confirme...
Table S1. Identify homology of CHS or CHS-like genes considered in Fig. 1. Table S2. Information ... more Table S1. Identify homology of CHS or CHS-like genes considered in Fig. 1. Table S2. Information of the CHS candidate genes and primers used for expression analysis. Table S3. Standard curve used in flavonoids detection. (DOCX 127 kb)
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