Suppression of tomato wilt by cell-free supernatants of Acinetobacter baumannii isolates from wild cacao from the Colombian Amazon.

Tomato vascular wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol) is one of the most limiting diseases of this crop. The use of fungicides and varieties resistant to the pathogen has not provided adequate control of the disease. In this study, siderophore-producing bacteria isolated from wild cocoa trees from the Colombian Amazon were characterized to identify prominent strategies for plant protection. The isolates were taxonomically classified into five different genera. Eight of the fourteen were identified as bacteria of the Acinetobacter baumannii complex. Isolates CBIO024, CBIO086, CBIO117, CBIO123, and CBIO159 belonging to this complex showed the highest efficiency in siderophore synthesis, producing these molecules in a range of 91-129 µmol/L deferoxamine mesylate equivalents. A reduction in disease severity of up to 45% was obtained when plants were pretreated with CBIO117 siderophore-rich cell-free supernatant (SodSid). Regarding the mechanism of action that caused antagonistic activity against Fol, it was found that plants infected only with Fol and plants pretreated with SodSid CBIO117 and infected with Fol showed higher levels of PR1 and ERF1 gene expression than control plants. In contrast, MYC2 gene expression was not induced by the SodSid CBIO117 application. However, it was upregulated in plants infected with Fol and plants pretreated with SodSid CBIO117 and infected with the pathogen. In addition to the disease suppression exerted by SodSid CBIO117, the results suggest that the mechanism underlying this effect is related to an induction of systemic defense through the salicylic acid, ethylene, and priming defense via the jasmonic acid pathway.

Complete genome sequence of Pediococcus acidilactici A40, a bacterium with biocontrol and plant growth-promoting properties.

We report the complete genome assembly of Pediococcus acidilactici A40, a bacterium with biocontrol and plant growth-promoting properties, obtained from Colombia.

Genome Sequence Data Reveal at Least Two Distinct Incursions of the Tropical Race 4 Variant of Fusarium Wilt into South America.

The global banana industry is threatened by one of the most devastating diseases: Fusarium wilt of banana. Fusarium wilt of banana is caused by the soilborne fungus Fusarium oxysporum f. sp. cubense (Foc), which almost annihilated the banana production in the late 1950s. A new strain of Foc, known as tropical race 4 (TR4), attacks a wide range of banana varieties, including Cavendish clones, which are the source of 99% of banana exports. In 2019, Foc TR4 was reported in Colombia, and more recently (2021) in Peru. In this study, we sequenced three fungal isolates identified as Foc TR4 from La Guajira (Colombia) and compared them against 19 whole-genome sequences of Foc TR4 publicly available, including four genome sequences recently released from Peru. To understand the genetic relatedness of the Colombian Foc TR4 isolates and those from Peru, we conducted a phylogenetic analysis based on a genome-wide set of single nucleotide polymorphisms (SNPs). Additionally, we compared the genomes of the 22 available Foc TR4 isolates, looking for the presence-absence of gene polymorphisms and genomic regions. Our results reveal that (i) the Colombian and Peruvian isolates are genetically distant, which could be better explained by independent incursions of the pathogen to the continent, and (ii) there is a high correspondence between the genetic relatedness and geographic origin of Foc TR4. The profile of present/absent genes and the distribution of missing genomic regions showed a high correspondence to the clades recovered in the phylogenetic analysis, supporting the results obtained by SNP-based phylogeny.

Close-Range Thermography and Reflectance Spectroscopy Support In Vitro and In Vivo Characterization of Colletotrichum spp. Isolates from Mango Fruits.

Colletotrichum causing anthracnose in mango is known for its variable virulence that may have an effect on disease development and efficacy of management strategies. In this study, we characterized Colletotrichum spp. isolated from mango fruits under in vitro and in vivo conditions using close-range thermography and reflectance spectroscopy. Twenty-six isolates were phylogenetically characterized to ascertain species using the internal transcribed spacer sequence. Virulence, spectral (in vivo and in vitro), and thermographic responses (in vivo) of these isolates were analyzed. Isolates were grouped into the Colletotrichum gloeosporioides species complex and classified into eight morphotypes. Mycelial growth, conidia production, sporulation abundance, and area under disease progress curve (AUDPC) varied largely among isolates. Disease symptoms were observed 4 days after inoculation (dai), and, for most morphotypes, changes in tissue temperature were registered at 11 dai, with the greatest decrease at 14 dai with pathogen sporulation. In vitro and in vivo morphotypes shared changes in the spectrum range, and main variations were found in the number of informative spectral bands. In vivo average gross reflectance was higher in disease-inoculated tissue than in healthy uninoculated tissue. Morphotype responses varied depending on AUDPC values and postinoculation time. Discriminant analysis of the spectral response using principal component analysis and partial least squares regression explained 94 to 96.3 and 98 to 99.9% of the variance from in vitro and in vivo tests, respectively. Spectral markers were obtained for four distinct morphotype groups. We found three (550 to 650, 650.1 to 790, and 1,300 to 1,400 nm) and two (520 to 830 and 1,100 to 1,450 nm) regions with highly (P < 0.05) discriminant spectral bands for diseased fruits and morphotype characterization.

Phenotypic and Genotypic Characterization of Phytophthora infestans Isolates Associated with Tomato and Potato Crops in Colombia.

Late blight disease, caused by the plant pathogen Phytophthora infestans, is one of the major threats for tomato and potato crops. Monitoring the populations of P. infestans is important to determine if there are changes in the sensitivity to fungicides and host preference. In this study, microsatellite markers and mitochondrial haplotypes were used to assess the genotype of isolates of P. infestans collected from tomato and potato plants in Colombia. Furthermore, sensitivity to the three fungicides cymoxanil (penetrant fungicide), mefenoxam, and fluopicolide (systemic fungicides), and tomato-potato host preference, were evaluated. Mitochondrial haplotyping showed that isolates collected on tomato were from the genetic groups Ia and Ib, while isolates collected on potatoes belonged to group IIa. Microsatellite analyses showed that isolates from tomato form two groups, including the Ib mitochondrial haplotype (which is genetically close to the US-1 clonal lineage) and the Ia haplotype (related to the EC-3 lineage), whereas Colombian isolates from potato formed a separate group. Furthermore, differences in sensitivity to fungicides were observed. Eighty-one percent of the isolates tested were resistant to mefenoxam with an EC50 >10 µg ml-1. Forty-two percent of the isolates showed an intermediate resistance to cymoxanil. The EC50 values ranged between 1 and 10 µg ml-1. For fluopicolide, 90% of the isolates were sensitive, with EC50 <1 µg ml-1. Host preference assays showed that potato isolates infected both host species. Thus, isolates that infect potatoes may pose a risk for tomato crops nearby.

Draft Genome Sequence and De Novo Assembly of a Fusarium oxysporum f. sp. lycopersici Isolate Collected from the Andean Region in Colombia.

We report a draft genome assembly of the causal agent of tomato vascular wilt, Fusarium oxysporum f. sp. lycopersici isolate 59, obtained from the Andean region in Colombia.

Efficacy of Disinfectants against Fusarium oxysporum f. sp. cubense Tropical Race 4 Isolated from La Guajira, Colombia.

Banana, the main export fruit for Colombia, is threatened by Fusarium wilt (FWB), caused by Fusarium oxysporum f. sp. cubense (Foc), tropical race 4 (TR4). Pathogen containment through disinfecting tools, machinery, shoes, and any means that may carry contaminated soil particles with proper disinfectants is at the forefront of disease management. In this study, the biocide efficacy of 10 commercial quaternary ammonium compounds (QACs) products and one based on glutaraldehyde (GA) were evaluated on both reproductive structures (microconidia and macroconidia) and survival spores (chlamydospores) of Foc TR4 (strain 140038) isolated from La Guajira, Colombia. QACs were evaluated at 1200 ppm and two exposure times: <1 and 15 min in the absence or presence of soil. For GA disinfectant, four different concentrations (500, 800, 1200, and 2000 ppm) were evaluated at both contact times in the presence of soil. In the absence of soil, all QACs showed 100% biocidal efficiency against microconidia, macroconidia, and chlamydospores at both <1 and 15 min. The presence of soil decreased the efficacy of disinfectants, but some of them, such as QAC3_1st, QAC7_4th, and QAC5_4th, showed 98%, 98%, and 100% efficacy against Foc TR4 chlamydospores, respectively, after <1 min of contact time. For instance, the GA-based disinfectant was able to eliminate all Foc TR4 propagules after 15 min for all concentrations tested.

Combining transcriptome analysis and GWAS for identification and validation of marker genes in the Physalis peruviana-Fusarium oxysporum pathosystem.

Vascular wilt, caused by the pathogen Fusarium oxysporum f. sp. physali (Foph), is a major disease of cape gooseberry (Physalis peruviana L.) in Andean countries. Despite the economic losses caused by this disease, there are few studies related to molecular mechanisms in the P. peruviana-Foph pathosystem as a useful tool for crop improvement. This study evaluates eight candidate genes associated with this pathosystem, using real-time quantitative PCR (RT-qPCR). The genes were identified and selected from 1,653 differentially expressed genes (DEGs) derived from RNA-Seq analysis and from a previous genome-wide association study (GWAS) of this plant-pathogen interaction. Based on the RT-qPCR analysis, the tubuline (TUB) reference gene was selected for its highly stable expression in cape gooseberry. The RT-qPCR validation of the candidate genes revealed the biological variation in their expression according to their known biological function. Three genes related to the first line of resistance/defense responses were highly expressed earlier during infection in a susceptible genotype, while three others were overexpressed later, mostly in the tolerant genotype. These genes are mainly involved in signaling pathways after pathogen recognition, mediated by hormones such as ethylene and salicylic acid. This study provided the first insight to uncover the molecular mechanism from the P. peruviana-Foph pathosystem. The genes validated here have important implications in the disease progress and allow a better understanding of the defense response in cape gooseberry at the molecular level. Derived molecular markers from these genes could facilitate the identification of tolerant/susceptible genotypes for use in breeding schemes.

A novel Transposable element-derived microRNA participates in plant immunity to rice blast disease.

MicroRNAs (miRNAs) are small non-coding RNAs that direct post-transcriptional gene silencing in plant development and stress responses through cleavage or translational repression of target mRNAs. Here, we report the identification and functional characterization of a new member of the miR812 family in rice (named as miR812w) involved in disease resistance. miR812w is present in cultivated Oryza species, both japonica and indica subspecies, and wild rice species within the Oryza genus, but not in dicotyledonous species. miR812w is a 24nt-long that requires DCL3 for its biogenesis and is loaded into AGO4 proteins. Whereas overexpression of miR812w increased resistance to infection by the rice blast fungus Magnaporthe oryzae, CRISPR/Cas9-mediated MIR812w editing enhances disease susceptibility, supporting that miR812w plays a role in blast resistance. We show that miR812w derives from the Stowaway type of rice MITEs (Miniature Inverted-Repeat Transposable Elements). Moreover, miR812w directs DNA methylation in trans at target genes that have integrated a Stowaway MITE copy into their 3' or 5' untranslated region (ACO3, CIPK10, LRR genes), as well as in cis at the MIR812w locus. The target genes of miR812 were found to be hypo-methylated around the miR812 recognition site, their expression being up-regulated in transgene-free CRISPR/Cas9-edited miR812 plants. These findings further support that, in addition to post-transcriptional regulation of gene expression, miRNAs can exert their regulatory function at the transcriptional level. This relationship between miR812w and Stowaway MITEs integrated into multiple coding genes might eventually create a network for miR812w-mediated regulation of gene expression with implications in rice immunity.

A new method for the inoculation of Phytophthora palmivora (Butler) into cacao seedlings under greenhouse conditions.

BACKGROUND: The black pod disease affects cacao plantations worldwide; it is caused by the oomycete species of the genus Phytophthora. The resistance of cacao plants to the black pod is commonly evaluated by artificial inoculation of the pathogen and the monitoring of the disease symptoms. However, it is difficult to identify resistant plants because the commonly used methods for the inoculation of the pathogens produce inconsistent results. Therefore, this study aimed to develop an efficient and reliable method to evaluate the resistance of Theobroma cacao seedlings to the infection by Phytophthora palmivora. RESULTS: Seedlings of different cacao genotypes were inoculated with P. palmivora under greenhouse conditions using the previously reported inoculation methods and a newly proposed method, the agar-water solution method. While none of the previously reported methods was effective, the agar-water solution method ensured a 100% seedling infection under greenhouse conditions. The proposed agar-water methodology is fast, simple and reproducible. Furthermore, the evaluation of this method in susceptible (CCN-51) and tolerant (SCA-6) T. cacao genotypes produced the expected contrasting results. CONCLUSIONS: The agar-water solution method presented in this study is an efficient alternative inoculation protocol for the identification of cacao genotypes that are resistant to black pod under greenhouse conditions.

Characterization of Pathogenic and Nonpathogenic Fusarium oxysporum Isolates Associated with Commercial Tomato Crops in the Andean Region of Colombia.

In Colombia, tomato production under protected conditions represents an important economic contribution to the agricultural sector. Fusarium wilt diseases, caused by pathogenic formae speciales of the soil-borne fungus Fusarium oxysporum Schltdl., cause significant yield losses in tomatoes throughout the world. Investigation of the F. oxysporum-tomato pathosystem in Colombia is required to develop appropriate alternative disease management. In this study, 120 fungal isolates were obtained from four different departments in the Central Andean Region in Colombia from tomato crops with symptoms of wilt disease. A molecular characterization of the fungal isolates was performed using the SIX1, SIX3, and SIX4 effector genes of Fusarium oxysporum f. sp. lycopersici W.C. Snyder & H.N. Hansen (Fol). Additionally, we developed a new specific marker to distinguish between Fusarium oxysporum f. sp. radicis-lycopersici Jarvis & Shoemaker (Forl) and Fol isolates. Furthermore, a phylogenetic analysis using the Translation Elongation Factor 1-alpha (EF1a) gene was performed with the collected isolates. Two isolates (named Fol59 and Fol-UDC10) were identified as Fol race 2, four isolates were identified as Forl, six isolates were identified as F. solani, and most of the isolates were grouped within the F. oxysporum species complex. The phylogenetic tree of EF1a showed that most of the isolates could potentially correspond to nonpathogenic strains of F. oxysporum. Additional pathogenicity assays carried out with Fol59 and Fol-UDC10 confirmed that both isolates were highly virulent strains. This study represents a contribution to the understanding of the local interaction between tomatoes and F. oxysporum in Colombia.

QTL analysis reveals quantitative resistant loci for Phytophthora infestans and Tecia solanivora in tetraploid potato (Solanum tuberosum L.).

Late blight and Guatemalan potato tuber moth caused by Phytophthora infestans and Tecia solanivora, respectively, are major phytosanitary problems on potato crops in Colombia and Ecuador. Hence, the development of resistant cultivars is an alternative for their control. However, breeding initiatives for durable resistance using molecular tools are limited due to the genome complexity and high heterozygosity in autotetraploid potatoes. To contribute to a better understanding of the genetic basis underlying the resistance to P. infestans and T. solanivora in potato, the aim of this study was to identify QTLs for resistance to P. infestans and T. solanivora using a F1 tetraploid potato segregant population for both traits. Ninety-four individuals comprised this population. Parent genotypes and their progeny were genotyped using SOLCAP 12K potato array. Forty-five percent of the markers were polymorphic. A genetic linkage map was built with a length of 968.4 cM and 1,287 SNPs showing good distribution across the genome. Severity and incidence were evaluated in two crop cycles for two years. QTL analysis revealed six QTLs linked to P. infestans, four of these related to previous QTLs reported, and two novel QTLs (qrAUDPC-3 and qrAUDPC-8). Fifteen QTLs were linked to T. solanivora, being qIPC-6 and qOPA-6.1, and qIPC-10 and qIPC-10.1 stable in two different trials. This study is one of the first to identify QTLs for T. solanivora. As the population employed is a breeding population, results will contribute significantly to breeding programs to select resistant plant material, especially in countries where P. infestans and T. solanivora limit potato production.

The Arabidopsis miR396 mediates pathogen-associated molecular pattern-triggered immune responses against fungal pathogens.

MicroRNAs (miRNAs) play a pivotal role in regulating gene expression during plant development. Although a substantial fraction of plant miRNAs has proven responsive to pathogen infection, their role in disease resistance remains largely unknown, especially during fungal infections. In this study, we screened Arabidopsis thaliana lines in which miRNA activity has been reduced using artificial miRNA target mimics (MIM lines) for their response to fungal pathogens. Reduced activity of miR396 (MIM396 plants) was found to confer broad resistance to necrotrophic and hemibiotrophic fungal pathogens. MiR396 levels gradually decreased during fungal infection, thus, enabling its GRF (GROWTH-REGULATING FACTOR) transcription factor target genes to trigger host reprogramming. Pathogen resistance in MIM396 plants is based on a superactivation of defense responses consistent with a priming event during pathogen infection. Notably, low levels of miR396 are not translated in developmental defects in absence of pathogen challenge. Our findings support a role of miR396 in regulating plant immunity, and broaden our knowledge about the molecular players and processes that sustain defense priming. That miR396 modulates innate immunity without growth costs also suggests fine-tuning of miR396 levels as an effective biotechnological means for protection against pathogen infection.

Transcriptomic and proteomic approach to identify differentially expressed genes and proteins in Arabidopsis thaliana mutants lacking chloroplastic 1 and cytosolic FBPases reveals several levels of metabolic regulation.

BACKGROUND: During the photosynthesis, two isoforms of the fructose-1,6-bisphosphatase (FBPase), the chloroplastidial (cFBP1) and the cytosolic (cyFBP), catalyse the first irreversible step during the conversion of triose phosphates (TP) to starch or sucrose, respectively. Deficiency in cyFBP and cFBP1 isoforms provokes an imbalance of the starch/sucrose ratio, causing a dramatic effect on plant development when the plastidial enzyme is lacking. RESULTS: We study the correlation between the transcriptome and proteome profile in rosettes and roots when cFBP1 or cyFBP genes are disrupted in Arabidopsis thaliana knock-out mutants. By using a 70-mer oligonucleotide microarray representing the genome of Arabidopsis we were able to identify 1067 and 1243 genes whose expressions are altered in the rosettes and roots of the cfbp1 mutant respectively; whilst in rosettes and roots of cyfbp mutant 1068 and 1079 genes are being up- or down-regulated respectively. Quantitative real-time PCR validated 100% of a set of 14 selected genes differentially expressed according to our microarray analysis. Two-dimensional (2-D) gel electrophoresis-based proteomic analysis revealed quantitative differences in 36 and 26 proteins regulated in rosettes and roots of cfbp1, respectively, whereas the 18 and 48 others were regulated in rosettes and roots of cyfbp mutant, respectively. The genes differentially expressed and the proteins more or less abundant revealed changes in protein metabolism, RNA regulation, cell signalling and organization, carbon metabolism, redox regulation, and transport together with biotic and abiotic stress. Notably, a significant set (25%) of the proteins identified were also found to be regulated at a transcriptional level. CONCLUSION: This transcriptomic and proteomic analysis is the first comprehensive and comparative study of the gene/protein re-adjustment that occurs in photosynthetic and non-photosynthetic organs of Arabidopsis mutants lacking FBPase isoforms.

Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana.

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin-Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1.

In planta gene expression analysis of Xanthomonas oryzae pathovar oryzae, African strain MAI1.

BACKGROUND: Bacterial leaf blight causes significant yield losses in rice crops throughout Asia and Africa. Although both the Asian and African strains of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), induce similar symptoms, they are nevertheless genetically different, with the African strains being more closely related to the Asian X. oryzae pv. oryzicola (Xoc). RESULTS: Changes in gene expression of the African Xoo strain MAI1 in the susceptible rice cultivar Nipponbare were profiled, using an SSH Xoo DNA microarray. Microarray hybridization was performed comparing bacteria recovered from plant tissues at 1, 3, and 6 days after inoculation (dai) with bacteria grown in vitro. A total of 710 bacterial genes were found to be differentially expressed, with 407 up-regulated and 303 down-regulated. Expression profiling indicated that less than 20% of the 710 bacterial transcripts were induced in the first 24 h after inoculation, whereas 63% were differentially expressed at 6 dai. The 710 differentially expressed genes were one-end sequenced. 535 sequences were obtained from which 147 non-redundant sequences were identified. Differentially expressed genes were related to metabolism, secretion and transport, pathogen adherence to plant tissues, plant cell-wall degradation, IS elements, and virulence. In addition, various other genes encoding proteins with unknown function or showing no similarity to other proteins were also induced. The Xoo MAI1 non-redundant set of sequences was compared against several X. oryzae genomes, revealing a specific group of genes that was present only in MAI1. Numerous IS elements were also found to be differentially expressed. Quantitative real-time PCR confirmed 86% of the identified profile on a set of 14 genes selected according to the microarray analysis. CONCLUSIONS: This is the first report to compare the expression of Xoo genes in planta across different time points during infection. This work shows that as-yet-unidentified and potentially new virulence factors are appearing in an emerging African pathogen. It also confirms that African Xoo strains do differ from their Asian counterparts, even at the transcriptional level.

Genomic comparison between Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola, using suppression-subtractive hybridization.

Xanthomonas oryzae pathovar oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) cause bacterial diseases in rice: leaf blight and leaf streak, respectively. Although both the Asian and the African strains of Xoo induce similar symptoms, they are genetically different, with the African Xoo strains being more closely related to the Asian Xoc. To identify the sequences responsible for differences between African and Asian Xoo strains and their relatedness to Xoc strains, a suppression-subtractive hybridization (SSH) procedure was performed, using the African Xoo MAI1 strain as a tester and the Philippine Xoo PXO86 strain and Xoc BLS256 strain as drivers. A nonredundant set of 134 sequences from MAI1 was generated. Several DNA fragments isolated by SSH were similar to genes of unknown function, hypothetical proteins, genes related to the type III secretion system, and other pathogenicity-related genes. The specificity of various fragments was validated by Southern blot analysis. SSH sequences were compared with several xanthomonad genomes. In silico analysis revealed SSH sequences as specific to strain MAI1, revealing their potential as specific markers for further epidemiological and diagnostic studies. SSH proved to be a useful method for rapidly identifying specific genes among closely related X. oryzae strains.

Análisis de expresión génica durante la respuesta de defensa de la yuca a la bacteriosis vascular (Añublo Bacteriano) / Gene expression analysis during cassava defense response to bacterial blight disease

La bacteriosis vascular de la yuca es una destructiva enfermedad en Sur América y África, causada por la bacteria Xanthomonas axonopodis pv. manihotis (Xam). Produce pérdidas entre el 12 por cien y 100 por cien de los cultivos. Algunos estudios se han realizado a nivel bioquímico y citoquímico para conocer las respuestas de defensa de la yuca a Xam; sin embargo,las bases moleculares de los mecanismos de defensa no han sido aún caracterizadas. Con el propósito de identificar genes diferencialmente expresados durante la respuesta de la planta al patógeno, se ha construido una librería sustractiva, usando el método de Sustracción Diferencial en Cadena (DSC), con 1536 clones de dos variedades resistentes(MBRA 685 y SG 107-35). De esta librería fueron seleccionados al azar 110 clones para ser secuenciados y realizar búsquedas de similitud en bases de datos públicas. El análisis de secuencia mostró 14 clones con similitud a genes previamente reportados como involucrados en procesos de defensa en plantas, 70 clones con similitud a genes de plantas sin función conocida o que no presentaron similitud, representando nuevos genes potencialmente involucrados en las respuestas de defensa de la yuca. Finalmente fueron construidos microarreglos de ADNc, usando los clones seleccionados de las librerías sustractivas para confirmar su expresión diferencial durante el desarrollo dela infección.