Exogenous methyl jasmonate induced cassava defense response and enhanced resistance to Tetranychus urticae.

Exogenous application of methyl jasmonate (MeJA) could activate plant defense response against the two-spotted spider mite (TSSM), Tetranychus urticae Koch, in different plants. However, whether MeJA can also serve as an elicitor in cassava (Manihot esculenta Crantz) remains unknown. In this study, induced defense responses were investigated in TSSM-resistant cassava variety C1115 and TSSM-susceptible cassava variety KU50 when applied with MeJA. The performance of TSSM feeding on cassava plants that were pre-treated with various concentrations of MeJA was first evaluated. Subsequently, the activities of antioxidative enzymes (superoxide dismutase and catalase), detoxification enzymes (glutathione S-transferase, cytochrome P450 and carboxylesterase) and digestive enzymes (protease, amylase and invertase) in TSSM were analyzed at days 1, 2, 4 and 8 post-feeding. The results showed that MeJA treatment can induce cassava defense responses to TSSM in terms of reducing egg production and adult longevity as well as slowing development and prolonging the egg stage. Noticeably, C1115 exhibited stronger inhibition of TSSM development and reproduction than KU50. In addition, the activities of all the tested enzymes were induced in both C1115 and KU50, the most in C1115. We conclude that exogenous methyl jasmonate can induce cassava defense responses and enhance resistance to TSSM.

Identification of cassava germplasms resistant to two-spotted spider mite in China: From greenhouse large-scale screening to field validation.

Introduction: Utilization of resistant germplasm is considered as an effective, economical and eco-friendly strategy for cassava pest management. Tetranychus urticae, known as the two-spotted spider mite (TSSM), is a devastating pest in Asian cassava planting countries as well as in China. However, the resistant levels of abundant cassava germplasms to TSSM remains largely unknown. Methods: To fill this knowledge gap, we conducted screening of 202 cassava germplasm for resistance to TSSM in China based on the classification of mite damage phenotype, under both greenhouse and field conditions. Results: The three rounds of large-scale greenhouse experiments had identified two highly resistant (HR) varieties (C1115 and MIANDIAN), five resistant (R) varieties (SC5, SC9, SC15, COLUMBIA-4D and LIMIN) and five highly susceptible (HS) varieties (KU50, BREAD, SC205, TMS60444 and BRA900), besides, these 'HR' and 'R' varieties would significantly repress the normal development and reproduction of TSSM. In addition, the 12 cassava varieties selected from the greenhouse screening were further subjected to consecutive five years of field validation at Danzhou, Wuming and Baoshan. The seven resistant varieties not only exhibited stable TSSM-resistance performance across the three field environments, but also possessed the same resistant levels as the greenhouse identification, while the resistant varieties SC5 was an exception, which was identified as moderate resistant in Baoshan, indicating the variety-environment interaction may affect its resistance. Furthermore, regional yield estimation suggested that the higher the resistance level was, the better capacity in reducing the yield losses. Discussion: This study demonstrated that the TSSM-resistant varieties could be considered as ideal materials in mite control or in future breeding programme of mite-resistant cassava plant.

Overexpression of leucoanthocyanidin reductase or anthocyanidin reductase elevates tannins content and confers cassava resistance to two-spotted spider mite.

The two-spotted spider mite (TSSM) is a destructive cassava pest. Intensive demonstration of resistance mechanism greatly facilitates the creation of TSSM-resistant cassava germplasm. Gene to metabolite network plays a crucial role in modulating plant resistance, but little is known about the genes and related metabolites which are responsible for cassava resistance to TSSM. Here, a highly resistant (HR) and a highly susceptible (HS) cassava cultivar were used, integrative and comparative transcriptomic and metabolomic analyses between these two cultivars after TSSM infestation revealed that several genes and metabolites were closely related and significantly different in abundance. In particular, the expression of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes showed a high positive correlation with most of the metabolites in the tannin biosynthesis pathway. Furthermore, transgenic cassava lines overexpressing either of the genes elevated tannin concentrations and conferred cassava resistance to TSSM. Additionally, different forms of tannins possessed distinct bioactivity on TSSM, of which total condensed tannins (LC50 = 375.68 mg/l) showed maximum lethal effects followed by procyanidin B1 (LC50 = 3537.10 mg/l). This study accurately targets LAR, ANR and specific tannin compounds as critical genes and metabolites in shaping cassava resistance to TSSM, which could be considered as biomarkers for evaluation and creation of pest-resistant cassava germplasm.

MePAL6 regulates lignin accumulation to shape cassava resistance against two-spotted spider mite.

Introduction: The two-spotted spider mite (TSSM) is a devastating pest of cassava production in China. Lignin is considered as an important defensive barrier against pests and diseases, several genes participate in lignin biosynthesis, however, how these genes modulate lignin accumulation in cassava and shape TSSM-resistance is largely unknown. Methods: To fill this knowledge gap, while under TSSM infestation, the cassava lignin biosynthesis related genes were subjected to expression pattern analysis followed by family identification, and genes with significant induction were used for further function exploration. Results: Most genes involved in lignin biosynthesis were up-regulated when the mite-resistant cassava cultivars were infested by TSSM, noticeably, the MePAL gene presented the most vigorous induction among these genes. Therefore, we paid more attention to dissect the function of MePAL gene during cassava-TSSM interaction. Gene family identification showed that there are 6 MePAL members identified in cassava genome, further phylogenetic analysis, gene duplication, cis-elements and conserved motif prediction speculated that these genes may probably contribute to biotic stress responses in cassava. The transcription profile of the 6 MePAL genes in TSSM-resistant cassava cultivar SC9 indicated a universal up-regulation pattern. To further elucidate the potential correlation between MePAL expression and TSSM-resistance, the most strongly induced gene MePAL6 were silenced using virus-induced gene silencing (VIGS) assay, we found that silencing of MePAL6 in SC9 not only simultaneously suppressed the expression of other lignin biosynthesis genes such as 4-coumarate--CoA ligase (4CL), hydroxycinnamoyltransferase (HCT) and cinnamoyl-CoA reductase (CCR), but also resulted in decrease of lignin content. Ultimately, the suppression of MePAL6 in SC9 can lead to significant deterioration of TSSM-resistance. Discussion: This study accurately identified MePAL6 as critical genes in conferring cassava resistance to TSSM, which could be considered as promising marker gene for evaluating cassava resistance to insect pest.

Reference Gene Selection for Analyzing the Transcription Patterns of Two Fatty Acyl-CoA Reductase Genes From Paracoccus marginatus (Hemiptera: Pseudococcidae).

Paracoccus marginatus (Hemiptera: Pseudococcidae), known as the papaya mealybug, could cause considerable yield loss of several plants. To date, there is no molecular-based study of P. marginatus. Fatty acyl-CoA reductases (FARs) are key enzymes involved in wax synthesis. In the present study, we cloned and characterized coding sequences (CDS) of two FAR genes from P. marginatus. The results showed that PmFAR1 and PmFAR2 CDS were 1,590 and 1,497 bp in length, respectively, and sequence analysis indicated that these two genes both had the conservative motifs belonging to FAR_C superfamily. Furthermore, seven candidate reference genes were analyzed for their expression stability by using common algorithms including comparative ΔCq method, geNorm, NormFinder, BestKeeper, and RefFinder. Eventually, ß-actin and GAPDH were the best reference genes in evaluating the expression of those two FAR genes. We found that PmFAR1 and PmFAR2 showed distinct expression patterns in different life stages. Moreover, the transcription of PmFAR1 and PmFAR2 in P. marginatus fed on resistant cassava cultivars was significantly lower compared with those fed on susceptible ones, indicating the potential function of FAR genes in cassava resistance to P. marginatus. The present study might help in better understanding the molecular mechanism of cassava resistance to mealybug.