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Pathogens rely on expression of host susceptibility ( S ) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas phaseoli pv. manihotis , the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a . In this work, we direct methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display decreased CBB symptoms while maintaining normal growth and development. This work therefore presents an epigenome editing approach useful for crop improvement. Activating the expression of host susceptibility ( S ) genes is one of the strategies plant pathogens employed to promote infection of their host. Here, the authors show that targeted methylation at the TAL20 effector binding element of the cassava SWEET10a gene lead to resistance to Xanthomonas phaseoli .

Societal Impact Statement Taro is a root crop with wide geographic range and high cultural significance to Indigenous peoples of Hawaiʻi and the Pacific. Taro Leaf Blight (TLB) is a worldwide disease of taro, and Hawaiian taro varieties exhibited little resistance to it. To improve TLB‐resistance, conventional breeding was conducted by hand‐pollination. Out of ~3500 taro seedlings, 18 varieties were selected and evaluated under non‐flooded conditions at four locations in Hawaiʻi over 5 years. Four varieties were identified as high yielding and disease‐resistant. Further, to prevent confusion of new taro varieties with traditional Hawaiian taro, we developed DNA fingerprints to differentiate them. Taken together, these results have provided information regarding new plant material and new tools for those interested in conservation and breeding across the Pacific. Summary Breeding and evaluation of taro (Colocasia esculenta (L.) Schott) was conducted in Hawaiʻi to increase resistance to Taro Leaf Blight (TLB), caused by oomycete pathogen Phytophthora colocasiae Racib., while maintaining high yields and desirable eating qualities. When breeding Indigenous crops such as taro, it is important to consider issues of Indigenous, intellectual property rights. In response to concerns by cultural practitioners about potential confusion of new taro genotypes with traditional landraces, we also sought to develop DNA fingerprints using single nucleotide polymorphisms (SNPs) to identify the new genotypes. Conventional breeding of taro was conducted by hand‐pollination of Hawaiian taro landraces with sources of TLB‐resistance from multiple countries. The most promising lines were multiplied and evaluated under upland (i.e., non‐flooded) conditions on four major Hawaiian Islands over 5 years. DNA was extracted from these new genotypes, analyses of SNPs conducted, and results compared to those of traditional Hawaiian landraces. Bayesian yield analysis of these multi‐year and multi‐location trials identified taro genotypes 1016‐003, 1016‐019, 1024‐215, and 1005‐084 as producing twice the fresh weight corm yield as control “Maui Lehua” and exhibiting disease‐resistance across years and environments. DNA fingerprints of new genotypes were able to differentiate them from traditional Hawaiian landraces that make up a portion of their parentage. Conventional breeding of taro in Hawaiʻi and multi‐year, multi‐location field trials have resulted in high yielding, disease‐resistant genotypes that maintain desirable qualities of some traditional Hawaiian landraces. DNA fingerprints of these new genotypes distinguish them from these traditional landraces, helping to preserve heritage germplasm. Taro is a root crop with wide geographic range and high cultural significance to Indigenous peoples of Hawaiʻi and the Pacific. Taro Leaf Blight (TLB) is a worldwide disease of taro, and Hawaiian taro varieties exhibited little resistance to it. To improve TLB‐resistance, conventional breeding was conducted by hand‐pollination. Out of ~3500 taro seedlings, 18 varieties were selected and evaluated under non‐flooded conditions at four locations in Hawaiʻi over 5 years. Four varieties were identified as high yielding and disease‐resistant. Further, to prevent confusion of new taro varieties with traditional Hawaiian taro, we developed DNA fingerprints to differentiate them. Taken together, these results have provided information regarding new plant material and new tools for those interested in conservation and breeding across the Pacific.

Thirty sweetpotato ( Ipomoea batatas var. batatas ) genotypes were evaluated for yield, resistances to weevil or nematode pests, and consumer acceptance across three field trials planted at Pepe‘ekeo, Hawai‘i Island between 2017 to 2020. At harvest, storage roots were graded according to market standards, followed by scoring for damage by sweetpotato weevil ( Cylas formicarius elegantulus ), rough sweetpotato weevil ( Blosyrus asellus ), or nematodes; namely root-knot nematode ( Meloidogyne spp.) or reniform nematode ( Rotylenchus reniformis ) in each market class. There were significant differences in marketable yields among accessions when data were analyzed statistically across all three field trials, as well as individually. ‘Regal’ (PI 566650) and ‘Sumor’ (PI 566657) were among the top three highest-yielding genotypes for Trials 1 and 2 (when no insecticides were applied to control weevils), and among the top six highest-yielding genotypes for the joint analysis across three trials. Significant differences among genotypes for combined sweetpotato weevil damage (incidence of sweetpotato weevil alone or incidence of both weevils together) were found in the joint analysis across three trials. ‘Regal’ was among the lowest seven genotypes for combined sweetpotato weevil damage, supporting earlier reports of its moderate resistance to this pest. In addition, two genotypes produced by the World Vegetable Center (Shanhua, Taiwan) (WT-320 and WT-108), were among the lowest genotypes for combined sweetpotato weevil damage, in agreement with earlier reports of their substantial resistance to this pest. Providing access to diverse germplasm will help farmers react to increasing pest pressure, while still allowing for high marketable yields. In addition, breeding and selection for pest resistance could be an important addition to integrated pest management of sweetpotato in Hawai‘i.

Provitamin A biofortification and increased dry matter content are important breeding targets in cassava improvement programs worldwide. Biofortified varieties contribute to the alleviation of provitamin A deficiency, a leading cause of preventable blindness common among pre-school children and pregnant women in developing countries particularly Africa. Dry matter content is a major component of dry yield and thus underlies overall variety performance and acceptability by growers, processors, and consumers. Single nucleotide polymorphism (SNP) markers linked to these traits have recently been discovered through several genome-wide association studies but have not been deployed for routine marker-assisted selection (MAS). This is due to the lack of useful information on markers’ performances in diverse genetic backgrounds. To overcome this bottleneck, technical and biological validation of the loci associated with increased carotenoid content and dry matter content were carried out using populations independent of the marker discovery population. In the present study, seven previously identified markers for these traits were converted to a robust set of uniplex allele-specific polymerase chain reaction (PCR) assays and validated in two independent pre-breeding and breeding populations. These assays were efficient in discriminating marker genotypic classes and had an average call rate greater than 98%. A high correlation was observed between the predicted and observed carotenoid content as inferred by root yellowness intensity in the breeding (r = 0.92) and pre-breeding (r = 0.95) populations. On the other hand, dry matter content-markers had moderately low predictive accuracy in both populations (r< 0.40) due to the more quantitative nature of the trait. This work confirmed the markers’ effectiveness in multiple backgrounds, therefore, further strengthening their value in cassava biofortification to ensure nutritional security as well as dry matter content productivity. Our study provides a framework to guide future marker validation, thus leading to the more routine use of markers in MAS in cassava improvement programs.

Turmeric (Curcuma longa) and related Curcuma species have been used traditionally in India, China, Hawaii, and other cultures for millennia. Today they are used around the world for spice, medicine, dye, and religious purposes. Recent biomedical studies have corroborated the long-known traditional medicinal values of turmeric and its constituent curcuminoid compounds, which have anti-inflammatory, antioxidant, and anticarcinogenic properties. As part of statewide research and extension efforts to support an expanding turmeric industry, we examined yield of 14 accessions across different climatic zones in Hawaii to observe and describe Genotype × Environmental influences. Fresh turmeric yield differed significantly among genotypes. The overall yields observed in this work ranged 11.3–57.22 t ha−1 and generally agree with those in the literature. Data from the different sites suggest that fertility and water management are able to mitigate moderate stress imposed by climate change within a certain range, but suboptimal temperatures associated with high elevation in the tropics (>1000 m) are an important driver of lower yields. This suggests that high yielding turmeric varieties may possess wide adaptability and may perform well across diverse environments. However, site-specific evaluations will still be necessary, particularly in environments outside turmeric’s environmental optima and in the presence of high pest pressure.

Tissue-cultured, virus-tested (TC) plantlets of sweetpotato ( Ipomoea batatas var. batatas ) cultivars Okinawan, LA 08-21p, and Murasaki-29 were obtained from Louisiana State University Agricultural Center. The objectives of field trials conducted at the Kula Agricultural Park, Maui, HI, were to compare yield and pest resistance of 1) ‘Okinawan’ obtained from a commercial (C) field with TC ‘Okinawan’ and 2) TC Okinawan with the aforementioned TC cultivars. Trials were planted Oct. 2015 and Aug. 2016 and harvested 5 months later. Storage roots were graded according to State of Hawai’i standards, and marketable yields included Grades AA, A, and B. In addition, injuries due to sweetpotato weevil ( Cylas formicarius elegantulus ) or rough sweetpotato weevil ( Blosyrus asellus ) were estimated. In both trials, fresh and dry weights of marketable storage roots of TC ‘Okinawan’ were nearly twice those from commercial planting material. In both trials, marketable fresh weights differed among the three TC cultivars; however, significant interactions were found, indicating that yields of cultivars differed between years. In the first field trial, ‘LA 08-21p’ had fresh marketable yields 1.6 to 1.7 times greater than TC ‘Okinawan’ and Murasaki-29, respectively. In the second trial, fresh marketable yields of TC ‘Okinawan’ and ‘LA 08-21p’were similar and 1.7 to 1.5 times greater than that of ‘Murasaki-29’, respectively. In both trials, ‘LA 08-21p’ had greater sweetpotato weevil injury than did the other two cultivars. Interestingly, in the second year, TC ‘Okinawan’ had greater rough sweetpotato weevil injury than did the other cultivars. Our results indicate that tissue-cultured planting materials increased marketable yields of TC ‘Okinawan’ compared with C ‘Okinawan’ sweetpotato and that the other TC cultivars did not produce greater yields than TC Okinawan.

Bacteria from the genus Xanthomonas are prolific phytopathogens that elicit disease in over 400 plant species. Xanthomonads carry a repertoire of specialized proteins called transcription activator-like (TAL) effectors that promote disease and pathogen virulence by inducing expression of host susceptibility (S) genes. Xanthomonas phaseoli pv. manihotis (Xpm) causes bacterial blight on the staple food crop, cassava. The Xpm effector, TAL20, induces ectopic expression of the S gene, MeSWEET10a, a sugar transporter that contributes to cassava bacterial blight susceptibility. We used CRISPR/Cas9 to generate multiple cassava lines with edits to the MeSWEET10a TAL20 effector binding site and/or coding sequence. In several of the regenerated lines, MeSWEET10a expression was no longer induced by Xpm and in these cases, we observed reduced cassava bacterial blight disease symptoms post Xpm infection. MeSWEET10a is expressed in cassava flowers. Therefore, we investigated flower development and reproductive function in an MeSWEET10a mutant line. We found that the MeSWEET10a mutant produced phenotypically wildtype cassava flowers and viable F1 seed. Thus, blocking MeSWEET10a induction is a viable strategy for decreasing cassava susceptibility to CBB.

Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas phaseoli pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. In this work, we direct methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display decreased CBB symptoms while maintaining normal growth and development. This work therefore presents an epigenome editing approach useful for crop improvement.Competing Interest StatementThe authors have declared no competing interest.Footnotes* This article has been updated to include more off-target analysis in addition to other, more minor, changes.

Provitamin A biofortification and increased dry matter content are important breeding targets in cassava improvement programs worldwide. Biofortified varieties contribute to the alleviation of provitamin A deficiency, a leading cause of preventable blindness in developing countries. Dry matter content is a major component of dry yield and thus underlies overall variety performance and acceptability by growers, processors, and consumers. SNP markers linked to these traits have recently been discovered through several genome-wide association studies but have not been deployed for marker-assisted selection (MAS). Assessment of marker performance in diverse genetic backgrounds is an important step towards their deployment for routine MAS. In the present study, seven previously identified markers for these traits were converted to a robust set of uniplex allele-specific PCR assays and validated in two independent pre-breeding and breeding populations. These assays were efficient in discriminating marker genotypic classes and had an average call rate greater than 98%. A high correlation was observed between the predicted and observed carotenoid content as inferred by root yellowness intensity in the breeding (r = 0.92) and pre-breeding (r = 0.95) populations. On the other hand, dry matter content-markers had moderately low predictive accuracy in both populations (r < 0.40) due to the more quantitative nature of the trait. This work confirmed marker effectiveness in multiple backgrounds, therefore, further strengthening its value in cassava biofortification to ensure nutritional security as well as dry matter content productivity. Our study provides a framework to guide future marker development, thus leading to more routine use of markers in MAS in cassava improvement programs. Competing Interest Statement The authors have declared no competing interest.