Received: 9 January 2021  |  Revised: 19 June 2021  |  Accepted: 21 June 2021 DOI: 10.1111/mpp.13107 R E V I E W Strategies to combat the problem of yam anthracnose disease: Status and prospects Valentine Otang Ntui1,2  | Edak Aniedi Uyoh1 | Effiom Eyo Ita1 | Aniedi-A basi Akpan Markson3 | Jaindra Nath Tripathi2  | Nkese Ime Okon1 | Mfon Okon Akpan1 | Julius Oyohosuho Phillip1  | Ebiamadon Andi Brisibe1 | Ene- Obong Effiom Ene- Obong1 | Leena Tripathi2 1Department of Genetics and Biotechnology, University of Calabar, Calabar, Nigeria Abstract 2International Institute of Tropical Yam (Dioscorea spp.) anthracnose, caused by Colletotrichum alatae, is the most devas- Agriculture, Nairobi, Kenya tating fungal disease of yam in West Africa, leading to 50%–9 0% of tuber yield losses 3Department of Plant and Ecological Studies, University of Calabar, Calabar, Nigeria in severe cases. In some instances, plants die without producing any tubers or each shoot may produce several small tubers before it dies if the disease strikes early. C. Correspondence Valentine Otang Ntui, Department of alatae affects all parts of the yam plant at all stages of development, including leaves, Genetics and Biotechnology, University stems, tubers, and seeds of yams, and it is highly prevalent in the yam belt region of Calabar, Calabar. P. M. B. 1115, Calabar, Nigeria. and other yam- producing countries in the world. Traditional methods adopted by Email: ntuival@yahoo.com; v.ntui@cgiar.org farmers to control the disease have not been very successful. Fungicides have also Funding information failed to provide long- lasting control. Although conventional breeding and genomics- Nigerian Tertiary Education Trust Fund assisted breeding have been used to develop some level of resistance to anthracnose (TETFund) in Dioscorea alata, the appearance of new and more virulent strains makes the devel- opment of improved varieties with broad- spectrum and durable resistance critical. These shortcomings, coupled with interspecific incompatibility, dioecy, polyploidy, poor flowering, and the long breeding cycle of the crop, have prompted research- ers to explore biotechnological techniques to complement conventional breeding to speed up crop improvement. Modern biotechnological tools have the potential of producing fungus- resistant cultivars, thereby bypassing the natural bottlenecks of traditional breeding. This article reviews the existing biotechnological strategies and proposes several approaches that could be adopted to develop anthracnose- resistant yam varieties for improved food security in West Africa. K E Y W O R D S anthracnose, CRISPR/Cas, Dioscorea spp., fungal diseases, genomics- assisted breeding, new breeding techniques, RNAi, yam This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd. 1302  |  wileyonlinelibrary.com/journal/mpp Mol Plant Pathol. 2021;22:1302–1314. NTUI eT al.      |  1303 1  | INTRODUC TION have shown evidence of severe anthracnose in D. rotundata in West Africa (Abang et al., 2003; Akem, 1999; Azeteh et al., 2019). Yam is an economically important, starchy staple for millions of peo- Combating the effects of this disease through application of ple in tropical and subtropical regions of the world. Presently, West fungicides is detrimental to the environment; it also adds to the al- Africa is responsible for approximately 95% of total global yam pro- ready high production cost and could induce development of resis- duction (FAOSTAT, 2018). The genus Dioscorea has over 600 species, tant strains of the pathogen (Onyeka et al., 2006). Cultural practices but only 11 are cultivated as food crops, which are Dioscorea alata such as crop rotation, destruction of infected plants, regular weed- (greater/water/winged yam; South-e ast Asia, Melanesia), Dioscorea ing, and planting of disease-f ree materials (Nwankiti & Arene, 1978) bulbifera (aerial/bulbil- bearing yam; South America, Africa, Asia, and have been used to manage the disease; however, the protection Melanesia), Dioscorea cayenensis (yellow Guinea yam; West Africa), offered by these methods is often insufficient and only temporary. Dioscorea dumetorum (sweet yam; West Africa), Dioscorea esculenta Host plant resistance is an environmentally friendly and sustainable (lesser/Asiatic yam; South- east Asia, Melanesia), Dioscorea japonica management strategy to control the disease (Onyeka et al., 2006) (glutinous/Japanese yam; Japan), Dioscorea nummularia (Pacific/ even though the transfer of desirable traits into the crop may be spiny yam; Melanesia), Dioscorea oppositifolia (Chinese yam; China), stalled by several factors with conventional breeding. Several Dioscorea pentaphylla (five-l eaved yam; South- east Asia, Melanesia), breeding programmes in India, Ivory Coast, Ghana, Guadeloupe, Dioscorea rotundata (white Guinea yam; West Africa), and Dioscorea Nigeria, and Vanuatu are making progress towards the production trifida (aja, aje, cush- cush, yampi; South America). However, some of an anthracnose- resistant hybrid (Lebot et al., 2019). However, a of the wild yam species are also important during food scarcity and multidisciplinary approach of traditional plant breeding, molecular are used for medicinal purposes (Govaerts et al., 2007; Tamiru et al., breeding, and new breeding techniques would be strategically ideal 2017; Verter & Vera Becvarova, 2015). Yam is rich in carbohydrate for developing new improved yam varieties to handle the challenge and thus provides a good source of energy. It also has unique medic- of variable anthracnose pathogens and accompanying pathogenicity. inal properties on account of its rich alkaloid content and steroidal Therefore, researchers could explore the development of improved compounds (Bantilan, 2019; Mignouna et al., 2008). Some yam spe- yam varieties with broad- spectrum and durable resistance by apply- cies are also used for the production of industrial starch. ing genetic engineering to complement conventional breeding. This Yam is the world's fourth most important tuber crop, after article reviews the potential tools that could be used to tackle yam cassava, potato, and sweet potato (IITA, 2013). For example, yam anthracnose in West Africa along with the associated problems and is grown in several countries in Central, East, and West Africa. future prospects. The yam belt region of West Africa, including Benin, Ghana, Ivory Coast, Nigeria, and Togo, is responsible for about 95% of the 72.6 million tonnes of global yam production (FAOSTAT, 2018). Nigeria 2  | ANTHR ACNOSE: C AUSAL AGENT, is presently the lead producer, contributing over 65% of global yam DISE A SE SYMPTOMS, DIAGNOSIS , AND production (FAOSTAT, 2018). Although different types of yams are SPRE AD grown globally, farmers in West Africa grow mainly D. rotundata, D. alata, and D. dumetorum. Colletotrichum is an economically important genus worldwide, Anthracnose or dieback disease of yam, which is caused pre- causing diseases in virtually all families of plants in the temperate, dominantly by Colletotrichum alatae (Weir et al., 2012), is the most tropical, and subtropical regions of the world (Bhunjun et al., 2021; widespread of all field diseases of yam, constituting a critical prob- Jayawardena et al., 2016b). It is rated as the eighth most important lem in all yam- producing areas of the world (Amusa et al., 2003). group of plant- pathogenic fungi worldwide. Members of the genus The effect is most severe during the rainy season. Young yam plants exist basically as pathogens but they have also been reported to lead are highly vulnerable, but infection can occur at all growth stages in endophytic and saprobic lifestyles (Hyde et al., 2014; Jayawardena susceptible yam genotypes and progress to severe disease, causing et al., 2016a). Colletotrichum is a complex genus with more than serious yield losses (Nwadili et al., 2017). Plants can be killed with- 1,000 described form-s pecies (Fokunang et al., 1995) and it is the out producing any tubers, or each shoot may produce several small sole member of the family Glomerellaceae (Maharachchikumbura tubers before it dies if the disease strikes early. Tuber yield losses et al., 2016). of up to 90% have been reported in susceptible genotypes in West Colletotrichum that infects yam is found in all yam- growing re- Africa (Akem, 1999; Mignucci et al., 1988). For example, up to 95% of gions and exhibits different growth characteristics. Based on the farmers in Ghana reported poor yield in D. alata due to anthracnose growth characteristics (growth rate, conidia, and appressorial disease. This poor yield negatively impacted their livelihoods as the morphology), four broad forms of Colletotrichum associated with farmers were unable to pay children's school fees and medical bills, yam anthracnose have been described (Abang et al., 2002). These service loans or purchase assets (Coffie, 2013). Among the culti- include the slow- growing grey (SGG), the fast- growing grey (FGG), vated species of yam, D. alata is the most susceptible to anthracnose the fast-g rowing salmon (FGS), and the fast- growing olive (FGO) (Amusa, 1997). However, contrary to the earlier reports of negligible forms. Of these, SGG is known to be the most aggressive and vir- attacks on other yam species (Mignucci et al., 1988), some reports ulent strain in terms of spread across the yam belt region, causing 1304  |     NTUI eT al. 100% defoliation and premature death of up to 76% of inoculated that spring from the primary hyphae and start colonizing the nearby plants (Abang et al., 2003; Mignouna et al., 2001). Some studies have cells. The process brings about development of visible lesions at shown that isolates of Colletotrichum from diseased yam leaves are the site of infection. Finally, conidia are formed on the surface of morphologically (Winch et al., 1984) and genetically (Abang et al., infected tissue and then they are dispersed by air currents, water 2002) diverse. However, these two studies used a broad species splash, and/or insects to start another infection cycle (De Silva et al., concept to group all isolates sourced from yam under the single 2019; Sharma & Kulshrestha, 2015). Symptoms start as pinpoint le- name C. gloeosporioides. Weir et al. (2012) found that a yam anthrac- sions of less than 2 mm on young yam leaves, frequently seen on the nose isolate from Nigeria together with those from Barbados (SAS8 adaxial surfaces. These lesions are dark brown or black on leaves, and SAS9), Guadeloupe (cgA13, GenBank accession GQ495617), and petioles, and stems, and are usually surrounded by a chlorotic halo, India (CBS 304.67, GenBank accession FJ940734) belonged to the which enlarges, coalesces, and eventually produces leaf necrosis same clade and matched the SGG group described by Abang et al. and stem dieback, with withered leaves and a scorched appearance (2002), and hence they were described as a distinct species named (Figure 1). Defoliation occurs in severe cases, leaving behind naked C. alatae (Weir et al., 2012). In July 2016, anthracnose- like lesions scorched vines. Death of plant cells due to toxic substances pro- were observed on the leaves of D. alata cultivar Da56 at a planta- duced by the fungus can permit other pathogens to colonize (Egesi tion in Danzhou City, Hainan Province, China. Morphological and et al., 2007). Some cultivars are reported to manifest leaf chlorosis molecular characteristics of the isolates matched descriptions of the and stunted growth, while others display leaf twists or folds leading SGG group observed in yam in West Africa (Lin et al., 2018). This iso- to stunted growth; these symptoms were credited to toxins from late was also referred to as C. alatae (Lin et al., 2018). While several the pathogen (Nwankiti & Ene, 1984). Depending on the symptoms authors (Bhunjun et al., 2021; Jayawardena et al., 2016c; Lin et al., on infected plants, anthracnose is labelled with various names, such 2018; Weir et al., 2012) have adopted the name C. alatae as causal as dieback, scorch, canker, Apollo, blight, and anthracnose/blotch agent of yam anthracnose, some authors (Kwodaga et al., 2020) have (Green & Simons, 1998; Winch et al., 1984). Anthracnose can be not picked up the name and are still using the name C. gloeosporioi- spread through dispersal of conidia by wind, rain, insects, and gar- des. These discrepancies may be due to the broad species concept of den tools, but it is mostly spread through rain splashes of the soil classifying all the forms into a single group. However, because SGG is containing spores on the plants. Anthracnose commences when the the most virulent form of anthracnose infecting yam in West Africa pathogen penetrates natural openings such as stomata and intact and several other regions, with no isolates from other hosts found in cuticle on the leaf surface (Nwankiti et al., 1987). Several authors the same clade (Weir et al., 2012), and based on recent reviews and (e.g., De Silva et al., 2017; Kolattukudy et al., 2000) have worked on studies of the genus (Bhunjun et al., 2021; Jayawardena et al., 2016c; the postinfection colonization strategies adopted by Colletotrichum Lin et al., 2018; Weir et al., 2012), in this work, the name C. alatae is species on fruits. However, little is known about the mechanism adopted as the causal agent of yam anthracnose. employed by the pathogen in colonizing yam leaf tissues. In the in- C. alatae attacks all parts of the yam plant, including leaves, tracellular hemibiotrophic phase, the pathogen produces primary stems, tubers, and seeds, at all stages of plant development (Abang hyphae and infection vesicles invading the epidermal and mesophyll et al., 2002) and it is present in the yam belt region and other yam- cells (De Silva et al., 2017). This early symptomless (biotrophic) phase producing countries in Africa. C. alatae begins its life cycle from ger- of infection has been reported in anthracnose infection of yam mination of conidia on the plant surface (leaf, stem, or tuber) forming (Jayawardena et al., 2016). This symptomless stage is followed by melanized infection structures called appressoria. This is followed the secretion of cell wall-d egrading enzymes that kill the host cells. by penetration of host tissue. Infection hyphae are formed in pri- Enzymes involved in the degradation of cell walls and maceration mary infected cells. This is the biotrophic stage of infection, where of yam leaf tissues have been shown to enhance yam leaf tissue no symptom is visible. The necrotrophic phase of infection follows, invasion by the pathogen (Amusa et al., 1993), besides phytotoxic which is characterized by the formation of thin secondary hyphae secondary metabolites (pathotoxins) implicated in further cellular F I G U R E 1   Symptoms of anthracnose in different yam tissues. (a– c) Symptoms in leaves. (d) Symptoms in yam tubers. (a) Pale yellow margins surrounding the lesions. (b,c) Dark brown spot dotting the leaf lamina. (d) Dark brown lesions on tubers. Arrows indicate the lesions NTUI eT al.      |  1305 destruction. The virulence of yam anthracnose pathogens is also screening (Infonet Biovision). Although it has been reported that the anchored on the ease of recombination of its virulence alleles and fungus can survive in an alternative host epiphytically (de Silva et al., its high potential for gene flow leading to genetic diversity between 2021), the host range of C. alatae needs to be determined as this is geographically distant populations. These potent genetic characters an important area open to investigation. Early planting allows for enhance its rapid evolution and aid its ability to influence disease quick plant canopy establishment before the months when rainfall, severity in a host cultivar (Abang et al., 2003). which favours disease proliferation, becomes severe (Egesi et al., The persistence of yam anthracnose in the farm is perpetuated 2007; Green, 1994). by potent sources of inoculum. The most important sources of The control of yam anthracnose has been accomplished mainly C. alatae inoculum are infested crop debris, infected tubers, and with chemical fungicides such as benomyl (benlate), maneb, chlo- alternate hosts (Amusa, 2000; Rojas et al., 2010). It has been shown rothalonil, and mancozeb, which require biweekly or monthly ap- that the pathogen is capable of being transmitted from foliage to plications. However, this could damage the environment, and its tuber, and from tuber to foliage the following season. Green (1994) frequent use could lead to the development of fungicide-r esistant has also reported that the pathogen overwinters in leaves, stems, strains (Onyeka et al., 2006). Fungicides can exert deleterious ef- and seeds. Weeds, such as Acalypha ciliata, Calapogonium mucunoi- fects, which can delay the onset of epidemics but cannot prevent des, Chromolaena odorata, Commelina spp., Euphobia heterophylla, them from developing during the rainy season. In general, copper Ipomoea triloba, and Spigelia anthelmia, have also been implicated as fungicides in combination with other fungicides such as mancozeb alternate hosts harbouring the pathogen populations of epidemic have been shown to be very effective against fungal pathogens proportion with cross- infection potential (Alleyne, 2001; Amusa, (Johnson & Hofman, 2009), but copper alone is less effective under 2000). The probable spread of anthracnose through spores has been high disease pressure and it is phytotoxic. However, it has been re- suggested through rain splash (Milgroom, 2003). ported that dithiocarbamet, mancozeb, and febran provide excellent Anthracnose can be diagnosed easily by the morphological ap- anthracnose control in the field (Akem, 2006). Thus, treating yam pearance of the disease on the tissues. Disease severity on leaves, tubers with fungicides such as benlate and captan has been reported lesion size, spore production on whole plants, and a pathogenicity to be effective in reducing fungal yam rot (Ogundana, 1971, 1981). test have been used as means to diagnose the disease. These meth- Benomyl was very efficient and applied only twice during the growth ods have shortcomings because according to Serra et al. (2011), dif- period but its application has been discontinued. The use of tecto ferent species of Colletotrichum are capable of infecting a single host, (thiabendazole) (Amusa & Ayinla, 1997; Ogundana, 1971, 1981), lo- making it difficult to differentiate in terms of their symptoms and cally made dry gin (Akinnusi et al., 1987; Ogali et al., 1991), or wood cultural morphology (Shi et al., 2008). Molecular techniques such as ash before storage (Osai, 1993), which are known to cause little or no PCR using species- specific internal transcribed spacers (ITSs) (ITS1 mammalian toxicity, has also been recommended. and ITS2) have been described as the best diagnostic tool for build- Nwankiti et al. (1987) reported that the most effective and desir- ing phylogenetic relationships in the Colletotrichum genus at the able means of controlling field yam diseases is by planting resistant species level (Chagas et al., 2017; Kwodaga et al., 2020; Serra et al., cultivars. Resistant yam cultivars could form the basis of sustainable 2011). Cloning and sequencing of PCR products, homology searches management strategies for anthracnose. Ongoing efforts aimed based on the available sequences in NCBI (Nwadili et al., 2017), in- at developing high-y ielding anthracnose- resistant yam cultivars formation from multilocus DNA analyses, and metabolic, proteomic, through classical breeding have been slow considering the biological morphological, physiological, biochemical, ecological, biogeograph- constraints related to the heterozygous and vegetative propagation ical, and mating data (Aime et al., 2021) will allow the identification of the crop. Despite its biological constraints, some progress has of pathogens at the species level. been reported in the development of anthracnose- resistant hybrids through conventional breeding (Lebot et al., 2019). Earlier investigations into the genetic control of the inheritance 3  | CONTROL AND MANAGEMENT OF of anthracnose in water yam showed that resistance is likely to be YAM ANTHR ACNOSE dominant and quantitatively inherited (Abang et al., 2001; Petro et al., 2011). Mignouna et al. (2001), using amplified fragment Cultural practices are geared towards the production of healthy length polymorphism (AFLP) markers, demonstrated that a single plants. Cultural practice is affordable and sustainable because it in- major dominant locus, designated as Dcg-1 , controls resistance in hibits the buildup of disease pathogens in the soil and plants. The the breeding line TDa 95/00328 and that this resistance is isolate- preventive measures usually have a long- term effect when compared specific. Subsequently, some yam cultivars resistant to anthracnose to other methods including the use of pesticides (OISAT, 2020). To have been identified. For example, TDa 1425 and TDr 2040 yam control yam anthracnose, disease avoidance is key. Practices that en- accessions in the collection of the Genetic Resource Unit of the courage disease avoidance include early planting, removal of plants International Institute for Tropical Agriculture (IITA) were found to that are alternative hosts for C. alatae, field sanitation, planting of be resistant to two isolates of the pathogen and were recommended healthy seed yams, intercropping with barrier plants, early stak- for use in areas endemic with yam anthracnose (Popoola et al., ing, adopting crop rotation (Jackson et al., 2000), and monitoring/ 2013). Also, D. alata lines Da. 110, Jas 2, and TCR 142 were found 1306  |     NTUI eT al. to be highly resistant to anthracnose following laboratory and field discovery could be useful in marker- assisted breeding in developing screening in India (Arya et al., 2019). These resistant lines were rec- resistance to anthracnose in D. alata. ommended for further evaluation and use in breeding programmes. SSR markers, due to their codominant nature, high level of Resistance to yam anthracnose reported in some yam cultivars by polymorphism, and high abundance, have been used to identify various authors was reported to be isolate- specific. Hopefully, a anthracnose- resistant and -s usceptible D. alata genotypes (Darkwa combination of both conventional and molecular techniques will be et al., 2020). Saski et al. (2015) developed 1,152 EST- SSRs from EST a better approach to develop yam cultivars with a wide range of sta- sequences generated from susceptible and resistant D. alata geno- ble resistance genes to protect against a broad spectrum of fungal types. In total, 388 of the EST-S SRs showed a polymorphism rate of pathogens for yam improvement. 34% for anthracnose on two different parental genotypes, indicating the possibility of using SSR to track anthracnose. Furthermore, they used genotyping by sequencing (GBS) tools such as EST sequencing, 4  | APPLIC ATION OF GENOMIC S- de novo sequencing, and GBS profiles and developed a comprehen- A SSISTED BREEDING AND HIGH- sive set of EST- SSRs, genomic SSRs, whole- genome single nucleotide THROUGHPUT PHENOT YPING FOR polymorphisms (SNPs), and reduced representation SNPs for resis- CONTROL OF YAM ANTHR ACNOSE tance to yam anthracnose in two D. alata genotypes, TDa95/00328 (resistant to anthracnose) and TDa95/310 (susceptible to anthrac- In recent years, genomics- assisted breeding (GAB), which integrates nose). However, the setback of the study is that many of the SNPs genomic tools with high- throughput phenotyping to improve crops, identified may be associated with broad- spectrum resistance or the has become a powerful plant breeding strategy. GAB uses molecular infection response (Darkwa et al., 2020). markers to facilitate the prediction of phenotype from a genotype Narina et al. (2011) identified genes differentially expressed in re- and allows breeders to select outstanding genotypes for further sponse to pathogen infection through a comparative transcriptomic evaluation. Molecular markers have been used to identify and locate analysis of infected susceptible (TDa95/0310) and two resistant yam genes and quantitative trait loci (QTLs) linked to disease resistance genotypes (TDa87/01091 and TDa95/0328). They generated 15,984 in several plants. The first trait mapping on disease resistance in yam ESTs in TDa95/0310; 15,196 ESTs in TDa95/0328; and 13,577 ESTs was done by Mignouna et al. (2002). The authors used a bulked seg- in TDa87/01091, with average sequence lengths of 411, 426, and regant analysis to detect rapid amplified polymorphic DNA (RAPD) 524 bases, respectively. TDa95/0328 and TDa87/01091 had 115 markers linked to yam mosaic virus (YMV) disease resistance in an and 180 highly expressed ESTs, respectively, which were found to F1 progeny resulting from a cross between a resistant male parent be linked to carbohydrate metabolism, cell wall biogenesis, lipid and (TDr89/0144) and a susceptible female parent (TDr87/00571). They amino acid metabolism, secondary and hormone metabolism, tran- identified a single locus linked to YMV resistance and named it Ymv- scription factors, protein synthesis, and signalling proteins as well 1. Also, they developed two RAPD markers closely linked to Ymv-1 as multiple pathogenesis-r elated and host defence- related genes in the same linkage group. These markers were successfully used (Darkwa et al., 2020). The highly expressed ESTs in the resistant gen- to identify Ymv-1 resistance in D. rotundata varieties and F1 prog- otypes could be responsible for their tolerance to the pathogen. The eny, indicating their possible utility in marker-a ssisted selection. limitation of this study is that the identified SNP markers may also be Furthermore, Mignouna et al. (2002) used QTL mapping to identify associated with other diseases and not anthracnose infection alone. one AFLP marker, E- 14/M52- 307, positioned on linkage group 2, that Screening of yam plants for anthracnose resistance, selecting was associated with anthracnose disease resistance. and segregating infected plants will reduce the spread of the dis- Petro et al. (2011) constructed an intraspecific genetic linkage ease. Considerable advances have been made in the development map of D. alata using 523 polymorphic AFLP markers and nine puta- of tools for the screening and detection of anthracnose. The IITA, tive QTLs on five linkage groups known for anthracnose resistance. Ibadan, Nigeria, through the AfricaYam project (https://africa yam. They noted that the phenotypic variance for each QTL was between org/the- africa yam-n ew- app- for- yam- anthr acnos e- disease) has de- 7.0% and 32.9%, and depending on the isolate and the variable con- veloped and standardized a detached leaf assay (DLA), the “Leaf sidered, the significant QTLs accounted for 26.4% to 73.7% of total Doctor” and “ESTIMATE,” for high- throughput screening of D. alata phenotypic variance. for anthracnose resistance (Kolade et al., 2018). The “ESTIMATE” Recently, Bhattacharjee et al. (2018) developed a genetic link- application uses yam anthracnose standard area diagrams for age map from 380 expressed sequence tag- simple sequence repeats image-b ased phenotyping in the field and DLA. The “Leaf Doctor” (EST-S SRs) on 20 linkage groups in order to identify QTLs controlling and “ESTIMATE” use artificial intelligence and machine learning to anthracnose resistance in D. alata. Linkage analysis conducted inde- accurately determine the percentage of leaf area affected by the pendently on data collected for 3 years by inoculating the mapping disease (Pethybridge & Nelson, 2015). This greatly enhances the population with the most virulent strain of the pathogen from West selection of promising lines for further evaluation (Kolade et al., Africa consistently found one QTL on linkage group 14. This QTL, 2018). This application is presently being adopted in different found at a position interval of 71.1– 84.8 cM, accounted for 68.5% countries to rapidly phenotype yam for anthracnose resistance of the total phenotypic variation in the average score data. This (Darkwa et al., 2020). NTUI eT al.      |  1307 5  | FUTURE PROSPEC TS OF APPLYING (Le et al., 2011). Also, transgenic tomato lines overexpressing a GENETIC ENGINEERING STR ATEGIES FOR wheat chitinase gene, chi194, under the control of the maize ubiq- CONTROL OF YAM ANTHR ACNOSE uitin 1 promoter were found to be highly resistant to Fusarium wilt disease of tomato caused by Fusarium oxysporum f. sp. lycoper- The use of conventional breeding to produce anthracnose- resistant sici (Girhepuje & Shinde, 2011). Mishra et al. (2016) demonstrated yam hybrids has shown some success. However, traditional breeding the control of guava wilt disease, caused by the soilborne fungus has not been exploited fully due to factors related to plant biology F. oxysporum f. sp. psidii, by expressing a Trichoderma endochitinase and low genetic variability. To overcome these hurdles, conventional gene in transgenic guava (Psidium guajava). As revealed by in vitro breeding needs to be complemented with genetic engineering, in- pathogen inhibition assays and spore germination assays, the crude cluding transgenic or genetic modification and genome editing tech- extract of the transformed plants inhibited the germination of fun- niques towards the improvement of the yam crop. These techniques gal conidia and plants were resistant to wilt disease. Expression of can be used to manipulate the different genotypes of yam without the wasabi defensin gene in melon via Agrobacterium transformation any barrier to produce improved varieties with broad- spectrum re- conferred resistance to Fusarium wilt and Alternaria leaf spot (Ntui sistance to anthracnose. The genetic engineering techniques allow et al., 2010). In a similar way, yam could be transformed with these altering the host-p lant genome either by manipulating the endog- genes or others used in different works to confer resistance to an- enous genes or by overexpressing transgenes from the same or dif- thracnose disease. Also, the overexpression of Dcg- 1 in susceptible ferent plant species or from microbes to confer resistance to fungal lines of yams can ultimately lead to the production of yam cultivars pathogens. Genes or molecules involved in defence signalling, de- resistant to anthracnose. This will boost efforts aimed at producing fence regulation, or other processes can easily be upregulated by anthracnose- resistant varieties of yam. transgenesis, enhancing resistance to anthracnose. The susceptibil- ity (S) genes, which function to facilitate anthracnose infection and colonization, can be silenced, resulting in enhanced resistance to 5.2 | RNAi strategy for engineering resistance the pathogen. Through genetic engineering, essential anthracnose against anthracnose genes can also be silenced through RNA interference (RNAi), result- ing in reduced disease incidence. For several fungal diseases, including anthracnose, Botrytis rots, downy and powdery mildews, and Fusarium wilts and rots, RNAi could be seen as a promising alternative to multiple control strat- 5.1 | Transgenic approaches through egies, including the use of fungicides, cultural practices, and the overexpression of fungal resistance genes deployment of resistant plant cultivars. To date, the feasibility of RNAi for targeted gene silencing via the exogenous addition of Several studies have developed systems for transient and stable synthetic double- stranded small interfering RNAs (siRNAs) tar- gene expression in yam, including particle bombardment (Tör et al., geting specific genes has been succinctly demonstrated in several 1993), polyethylene glycol (PEG)- mediated transfection of proto- fungi. For example, a considerable advance could be made using plasts (Tör et al., 1998), and Agrobacterium- mediated transformation RNAi technology in the fight against anthracnose that is caused by (Nyaboga et al., 2014; Quain et al., 2011). Among these protocols, several genera of ascomycete fungi including Colletotrichum linde- Agrobacterium-m ediated transformation is the most preferred be- muthianum, which adversely affects the yield of Phaseolus vulgaris cause it is easily available, facilitates the integration of large nu- (de Lima Castro et al., 2017; LeClair et al., 2015), Colletotrichum cleotide segments with negligible rearrangements, allows for the sublineola affecting sorghum, Colletotrichum gloeosporioides affect- transfer of only a single copy of the gene, and is relatively cheap. ing chilli and tomato (Mahto et al., 2020), and C. alatae causing yam A protocol for Agrobacterium-m ediated genetic transformation dieback and being the most serious disease affecting Dioscorea spe- of yam has been documented (Nyaboga et al., 2014). The authors cies (Figure 1), especially under intensive cultivation in the tropics transformed two cultivars of D. rotundata with Agrobacterium tume- (Ripoche et al., 2008). RNAi posttranscriptional gene silencing can faciens harbouring binary vectors containing gus and gfp as reporter be programmed with 21– 25-n ucleotide duplexes of siRNAs and long genes and obtained a transformation efficiency ranging from 9.4% double- stranded RNAs (dsRNAs) corresponding to different se- to 18.2%, depending on the cultivars, selectable marker genes, and quences in order to induce an effective antifungal response against the Agrobacterium strain used for transformation. This protocol the replication of the fungal pathogen, both in in vitro cultures as could be used to overexpress transgenes for anthracnose resist- well as during infection in the field (Machado et al., 2018; Wani ance. Although no work has documented the expression of foreign et al., 2010). It is possible that, as observed naturally in many eu- genes in yam for fungal resistance, several genes conferring resist- karyote kingdoms (Baulcombe, 2015), for example in the nematode ance to fungal diseases have been identified and expressed in other Caenorhabditis elegans (Fire et al., 1998), following the microinjection crops. For example, transgenic grapevine lines overexpressing a Vitis of 500–7 00- nucleotide siRNAs, RNAi may function as an adaptive, vinifera NPR1.1 gene developed via Agrobacterium- mediated trans- nucleic acid- based defence system in anthracnose-s usceptible crop formation were reported to be resistant against powdery mildew species by either inhibiting replication at different stages in the life 1308  |     NTUI eT al. cycle of the particular Colletotrichum pathogen, or simply by acting additional successes will come from a more detailed understand- as a retrotransposon silencing mechanism. However, irrespective of ing of the practical implications of using RNA-b ased therapeutics the exact gene silencing pathway(s) in use, there are several proofs through the production of dsRNA nucleotide intermediates against of principle regarding the feasibility of RNAi in the downregulation some prime target genes that can be used as therapy against differ- of specific genes in fungi (Li et al., 2010) including Neurospora crassa ent fungal diseases, especially anthracnose, which can be considered (Fulci & Macino, 2007; Romano & Macino, 1992), Mucor circinelloides as a paradigm shift in crop protection. (Billmyre et al., 2013), and Saccharomyces cerevisiae (Billmyre et al., 2013; Trieu et al., 2015). Usually, RNAi is initiated following the entry of a long dsRNA 5.3 | CRISPR/Cas9 gene editing strategy to develop such as an introduced transgene, a rogue genetic element, or a mi- anthracnose resistance crobial intruder into the cell, which triggers the RNAi pathway and results in the recruitment of the enzyme Dicer. Thereafter, the Dicer Recent advances in genome editing technologies using site- directed enzyme cleaves the dsRNA into 21– 25- nucleotide siRNA duplexes. nucleases (SDNs), such as meganucleases, encoded by mobile ge- Subsequently, an RNA- induced silencing complex (RISC) then distin- netic elements or introns, zinc- finger nucleases derived from guishes between the two siRNA strands as either sense or antisense eukaryotic Cys2His2 zinc finger proteins covalently linked to the nu- strands. While the sense strand is degraded, the antisense strand clease domain of the type IIS restriction enzyme Fok1, transcription is loaded into the RISC, which is used as guide to target mRNAs activator-l ike effector nucleases (TALENs) from TALEs of bacteria in a sequence-s pecific manner. Messenger RNAs, which code for Xanthomonas linked with the Fok1 nuclease domain, and CRISPR/ proteins, are then cleaved by the RISC. The activated RISC can re- Cas from the adaptive immunity system of Streptococcus pyogenes peatedly participate in mRNA degradation, thus inhibiting protein have enabled plant scientists to manipulate desired genes in crop synthesis in the cell. On account of this posttranscriptional gene plants (Tripathi et al., 2020). silencing mechanism, it could be speculated that specific dsRNAs Among these nucleases, CRISPR/Cas9 is the most powerful and might serve as promising vehicles that can be developed into mo- versatile tool for crop genome editing because of its simplicity, de- lecular tools for genetic improvement of food crops against fungal sign flexibility, and high efficiency and its ability to simultaneously infections, including anthracnose disease in yam. edit multiple genes (Ntui et al., 2020; Tripathi et al., 2019). The Based on reports, as might be expected, RNAi is astounding, CRISPR/Cas9 editing system consists of two basic components: the having the power to overwhelm fungal pathogens by turning off guide RNA (gRNA) and the Cas9 nuclease (Figure 2). Cas9 exhib- or silencing harmful genes (Almeida & Allshire, 2005; de Bakker its nuclease activity, recognizing target DNA by gRNA–D NA pairing et al., 2002). This is best illustrated by data from several recent in- between the 5′ leading sequence of gRNA. It also recognizes the vestigations. It has also been reported that barley and wheat plants protospacer adjacent motif (PAM) sequence and starts editing up- could be transgenically engineered to express dsRNAs that target stream of the sequence. The PAM is a trinucleotide sequence, usu- transcripts of the virulence factor Avra10 in the fungus Blumeria gr- ally NGG or NAG (Figure 2), where N can be any nucleotide, and aminis, which resulted in reduced powdery mildew infections in the serves as a recognition segment for Cas9 to start editing upstream. crops. Furthermore, it has been demonstrated in numerous studies The gRNA consists of a scaffold and a user- defined spacer sequence that host immune gene silencing is an effective approach in the con- (c.20 nucleotides) for genomic sequence targeting. It directs the trol of a wide range of taxonomically unrelated filamentous fungal Cas9 to induce precise double-s tranded breaks (DSBs), which are and oomycete pathogens (Machado et al., 2018). Aside from these, repaired either through the nonhomologous end-j oining (NHEJ) several studies using long, corresponding fragments of dsRNA also DNA repair pathway at the target site or homology-d irected repair showed that fungal replication can be inhibited in both Botrytis cine- (HDR), resulting in small insertions/deletions (indels) or substitution rea and Fusarium graminearum (Koch et al., 2013; Wang et al., 2016). of nucleotides. Of paramount significance and fascination in these studies was Based on the type of repair, the editing can be performed by the finding that the response, ascribed to posttranscriptional si- SDN1, SDN2, or SDN3 (Modrzejewski et al., 2019). SDN1 is based lencing of specific genes, showed exogenous transfected siRNAs on NHEJ resulting in random mutations in the host genome, causing and long dsRNAs that direct the sequence- specific degradation of gene silencing, gene knockout, or alteration in the gene function. In mRNAs encoding cognate receptors through which the fungal cells SDN2, a repair template identical to the DSB is added. The DSB is gain entry into plant cells (Machado et al., 2018). Collectively, these then repaired via HDR, resulting in nucleotide substitution or tar- observations are highly encouraging and could be of immense signif- geted indels. In SDN3, the DSB is repaired via HDR using a repair icance in the control of fungal diseases, including anthracnose. They template, which is longer than the homologous sequences in which illustrate the fact that using intracellular RNA- based therapeutics, the DSB is made, leading to the targeted insertion of foreign genes. developed against the right target gene(s) such as the fungal Dicer- The CRISPR/Cas system has become the method of choice for like 1 (DCL1) and DCL2 genes (Wang et al., 2016), RNAi might serve the control of fungal diseases in plants. So far, no work has been doc- as an effective strategy for developing a durable therapy against umented using CRISPR/Cas9 to develop resistance to anthracnose anthracnose in food crops including yam species. It is likely that in yam; however, the system has been used to induce resistance to NTUI eT al.      |  1309 F I G U R E 2   Schematic representation of the CRISPR/Cas9 gene editing mechanism. gRNA directs Cas9 to cleave the target sequence upstream of the protospacer adjacent motif (PAM), producing a double- stranded break (DSB). The DSB is subsequently repaired either by nonhomologous end- joining (NHEJ) or by homology- directed repair (HDR). Repair via NHEJ produces indels (knockout), whereas repair through HDR results in knockin fungal diseases in some plants species which could be applicable albino phenotype, and gene knockout was confirmed by sequence to yam. Fungal resistance via CRISPR/Cas9 was mainly achieved analysis (Syombua et al., 2021). This protocol will facilitate genome until now by targeting susceptibility (S) genes as well as ethylene- editing of yam targeting genes involved in resistance to anthracnose responsive factors (Das et al., 2019; Tripathi et al., 2019). During as well as other fungal diseases. pathogen invasion, S genes are activated by the pathogen to facili- tate pathogen growth and symptom development (Boch et al., 2014; van Schie & Takken, 2014). Editing of S genes has been reported to 6  | CHALLENGES IN DE VELOPING confer resistance to the corresponding pathogen, and in some cases ANTHR ACNOSE- RESISTANT YAM THROUGH even broad- spectrum resistance (Blanvillain- Baufumé et al., 2017; GENETIC ENGINEERING Kim et al., 2019; Olivia et al., 2019; Peng et al., 2017). For example, the mildew resistance locus O (MLO) is the most widely known S Management of anthracnose through cultural practices and produc- gene locus (Das et al., 2019). MLO encodes a seven- transmembrane tion of anthracnose- resistant yam varieties through conventional domain calmodulin- binding protein located at the plasma membrane breeding has shown some success. GAB has been used to create (Kim et al., 2002). Its role in susceptibility toward powdery mildew some levels of resistance, but the evolution of new ecotypes of the disease in monocot and dicot plants has also been confirmed (Kusch fungus requires the exploration of new breeding techniques for de- & Panstruga, 2017). Editing of MLO in wheat (Wang et al., 2014) veloping broad-s pectrum and durable resistance in yam against fun- and tomato (Nekrasov et al., 2017) conferred resistance to pow- gal pathogens. dery mildew. The MLO gene, together with other S genes identified Biotechnological approaches such as overexpression of fungus in other crops such as enhanced disease resistance 1 (EDR1) (Zhang resistance genes, RNAi, and genome editing require an efficient yam et al., 2017) and ethylene- responsive factor (ERF) (Santillán- Martínez genetic transformation protocol. Currently, yam genetic transfor- et al., 2020; Wang et al., 2016) would be an excellent candidate for mation is still a bottleneck that could hinder the rapid production developing resistance to yam anthracnose and other fungal diseases. of anthracnose-r esistant yam cultivars in West Africa. In a recently In yam, the CRISPR/Cas9- based genome editing has been docu- published article on genome editing of yam (Syombua et al., 2021), mented targeting the phytoene desaturase (PDS) gene (Syombua et al., only six transgenic events were recovered from several hundreds of 2021). PDS is a key enzyme in the carotenoid biosynthesis pathway, explants cocultivated with Agrobacterium. This indicates that yam catalysing the desaturation of phytoene into lycopene. PDS is often genetic transformation is still a major challenge. Developing an ef- used as a marker to establish genome editing in plants because its ficient yam transformation protocol, at least for the cultivars pre- disruption affects photosynthesis and gibberellin and carotenoid ferred by farmers, is thus a necessary requirement for improving biosynthesis, causing albinism and dwarfing, phenotypes that are yam, not just for anthracnose resistance but also for other agronom- easy to see. Mutated yam plantlets generated by Agrobacterium- ically important traits. Yam breeders/biotechnologists could explore mediated transformation showed either a partial or a complete the possibilities of using alternative ways to deliver transgenes to 1310  |     NTUI eT al. yam cells in order to bypass the laborious work of tissue culture. RNPs mutate the target sites immediately after delivery and then Methods such as agroinfiltration or in planta transformation could get rapidly degraded by endogenous proteases, leaving no traces of be adopted. Viral vector- based platforms for rapid and efficient de- foreign DNA elements. The mutant genotypes will be accepted with- livery of overexpression, RNAi, and CRISPR/Cas9 constructs could out any major regulatory issues. The use of more genomic tools will be adopted. YMV may be an excellent candidate for technologies contribute to the production of anthracnose- resistant genotypes focused on viral vectors. Like the tobacco rattle virus (TRV) vector, and help to overcome the regulatory challenges of classical genetic which has been commonly used as a vector to alter plants, YMV engineering and genome editing. may be genetically engineered to bear plasmids for onward trans- mission to yam cells through agroinfiltration (Liu et al., 2002; Ntui et al., 2013). Much like TRV, YMV is an RNA virus, making it a useful 7  | CONCLUSION candidate for viral- based transformation. Dioscorea alata bacilliform virus (DaBV) is another potential virus that could be adapted to bear Yam is an important staple food crop in West Africa and plays key plasmids. DaBV is a badnavirus that is known to incorporate into the roles in income generation and the sociocultural life of smallholder genome of the host and cause symptoms under conditions of stress. farmers. Anthracnose is the most important fungal disease affecting DaBV is thus an excellent candidate for modification as a vector to yam production and causing severe economic hardship to yam pro- deliver CRISPR/Cas9, RNAi, and overexpression constructs into yam ducers. The production of yam resistance to anthracnose by cultural cells. The modification of yam- based viruses as vectors could facili- practices and conventional breeding is a major challenge. The use tate the production of anthracnose-r esistant yam by agroinfiltration. of anthracnose- resistant yam varieties is the most sustainable way In planta transformation should be tried to rapidly develop re- of reducing losses due to this fungus. The development of durable sistance to anthracnose. In planta transformation involving a direct anthracnose- resistant varieties through conventional breeding using transformation of plant parts has been established as an innovative resistant germplasm from Asia and adoption of GAB and modern and simple technique for plant transformation. Whole yam plants, biotechnological tools will speed up the production of anthracnose- shoot tips, floral parts, or female reproductive parts such as zygotes, resistant yam varieties. The development of a robust genetic trans- embryos, and seeds should be exploited and optimized for in planta formation protocol and in planta transformation techniques, as well transformation of yam. This will overcome the challenges of trans- as agroinfiltration protocols using a viral vector-b ased platform, will formation and the problem of tissue culture- induced genetic vari- facilitate the production of anthracnose-r esistant yam cultivars. The ability in the transformants. availability of whole- genome sequence information of yam will enable The development of yam cultivars resistant to anthracnose the identification and editing of S genes conferring resistance to fun- through overexpression of a single antimicrobial gene may result gal pathogens. These technologies, if developed, could facilitate the in partial resistance and, in some cases, in resistance breakdown. production of anthracnose- resistant yam cultivars and hence increase Therefore, pyramiding (stacking) of some genes conferring resis- food security and income generation for yam farmers in West Africa. tance to fungal diseases might play a vital role in providing long- lasting resistance to anthracnose. Cotransformation or the use of a ACKNOWLEDG EMENTS marker- free approach will promote the stacking of fungus resistance The authors wish to thank the Tertiary Education Trust Fund genes in yam. (TETFund) of Nigeria for their financial support. This work was Successful application of CRISPR/Cas9-b ased genome editing to supported with funds from a research grant on “Production of induce resistance to anthracnose will require the availability of well- anthracnose- resistant yam seedlings for use by Nigerian farm- annotated genome sequences. With the recently developed genome ers” awarded to the Department of Genetics and Biotechnology, editing protocol for yam and the available whole- genome sequence University of Calabar, Calabar, Nigeria, by the TETFund. of D. rotundata and D. alata, S gene sequences known to confer re- sistance to fungal diseases in other crops could be identified and CONFLIC T OF INTERE S T edited in yam. Multiplexing of two or more of such genes may result The authors declare that no conflicts of interest exist. in durable resistance to anthracnose. Generation of anthracnose-r esistant yam either by classical AUTHOR CONTRIBUTIONS genetic engineering or genome editing will be regulated in some E.A.U. and E.E.E. conceived the original concept. E.A.U., V.O.N., countries in West Africa and may reduce its acceptability. Yam is veg- E.E.I., A.A.M., J.N.T., N.I.O., M.O.A., J.O.P., E.A.B., L.T., and E.E.E. etatively propagated and even with CRISP/Cas9- mediated plasmid wrote the manuscript. V.O.N., J.O.P., E.E.I., and J.N.T. prepared the delivery, transgenes cannot be removed by segregation as in sexually figures. propagated crops. This will be a major limitation of using these tech- nologies to develop anthracnose- resistant yam genotypes. 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