The Adsorption of a Polygalacturonase Isoenzyme of Botryodiplodia theobromae on Plant Tissues and the Implication on the Pathogenicity of the Fungus

The polygalacturonase isoeazyme (PG 3) of Botryodiplodia theobromae extracted from rotted sweet potato was adsorbed by sweet potato, potato, carrot, bean stem and tomato fruit to various degrees. Adsorption was greater with sweet potato and tomato fruit tissues. Carrots, bean stem and potato absorbed the enzyme to more or less the same degree. The enzyme was not adsorbed on tomato stem. A spore/mycelial suspension of B. theobromae infected the test tissues to various degrees. The enzyme completely macerated sweet potato roots, potato tubers and tomato fruits within 5 h while the bean stem and onion tissues were little affected by the enzyme. The tomato stem was neither infected by the fungus nor macerated by the enzyme.     

Role of Surface Substances on Germinated Spores of Ceratosystis fimbriata,Black Rot Fungus,in Host-parasite Interaction

Surface substances were isolated by sonication from the germinated spores of various strains of Ceratocystis fimbriata and characterized in relation to host-parasite specificity. The substances from the sweet potato strain, compatible with sweet potato, potently inhibited the spore agglutination of various strains by spore-agglutinating factor from sweet potato roots, while the substances from incompatible strains, that is, coffee, taro, and almond strains, weakly inhibited this agglutination. The substances from the sweet potato strain increased ethylene production from sweet potato roots infected by all strains tested, sweet potato, coffee, taro, and almond strains, which was possibly an index of pathogenicity. On the other hand, the substances from incompatible strains, coffee, taro, and almond strains, suppressed the ethylene production from the tissue infected by all four strains except the substances from almond strains on almond strain. Heat and trypsin treatments inactivated the spore agglutination inhibitory activity of the surface substances. Coincidently, these treatments extinguished the effect of the surface substances on pathogenicity of C. fimbriata on sweet potato roots.     

Resistance to sweet potato virus disease (SPVD) in wild East African Ipomoea

Sweet potato virus disease (SPVD), the most harmful disease of sweet potatoes in East Africa, is caused by mixed infection with sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Wild Ipomoea spp. native to East Africa (J cairica, I. hildebrandtii, I. involucra and J wightii) were graft-inoculated with SPVD-affected sweet potato scions. Inoculated plants were monitored for symptom development and tested for SPFMV and SPCSV by grafting to the indicator plant J setosa, and by enzyme-linked immunosorbent assay (ELISA). Virus-free scions of sweet potato cv. Jersey were grafted onto these wild Ipomoea spp. in the field, and scions collected 3 wk later were rooted in the greenhouse and tested for viruses using serological tests and bioassays. In all virus tests, J cairica and J involucra were not infected with either SPFMV or SPCSV. J wightii was infected with SPFMV, but not SPCSV, in the field and following experimental inoculation; J hildebrandtii was infected with SPCSV, but not SPFMV, following experimental inoculation. These data provide the first evidence of East African wild Ipomoea germplasm resistant to the viruses causing SPVD.     

Synergistic interactions of begomoviruses with Sweet potato chlorotic stunt virus (genus Crinivirus) in sweet potato (Ipomoea batatas L.)

Three hundred and ninety‐four sweet potato accessions from Latin America and East Africa were screened by polymerase chain reaction (PCR) for the presence of begomoviruses, and 46 were found to be positive. All were symptomless in sweet potato and generated leaf curling and/or chlorosis in Ipomoea setosa. The five most divergent isolates, based on complete genome sequences, were used to study interactions with Sweet potato chlorotic stunt virus (SPCSV), known to cause synergistic diseases with other viruses. Co‐infections led to increased titres of begomoviruses and decreased titres of SPCSV in all cases, although the extent of the changes varied notably between begomovirus isolates. Symptoms of leaf curling only developed temporarily in combination with isolate StV1 and coincided with the presence of the highest begomovirus concentrations in the plant. Small interfering RNA (siRNA) sequence analysis revealed that co‐infection of SPCSV with isolate StV1 led to relatively increased siRNA targeting of the central part of the SPCSV genome and a reduction in targeting of the genomic ends, but no changes to the targeting of StV1 relative to single infection of either virus. These changes were not observed in the interaction between SPCSV and the RNA virus Sweet potato feathery mottle virus (genus Potyvirus), implying specific effects of begomoviruses on RNA silencing of SPCSV in dually infected plants. Infection in RNase3‐expressing transgenic plants showed that this protein was sufficient to mediate this synergistic interaction with DNA viruses, similar to RNA viruses, but exposed distinct effects on RNA silencing when RNase3 was expressed from its native virus, or constitutively from a transgene, despite a similar pathogenic outcome.     

Induction of Furano-terpene Production and Formation of the Enzyme System from Mevalonate to Isopentenyl Pyrophosphate in Sweet Potato Root Tissue Injured by Ceratocystis fimbriata and by Toxic Chemicals

When sweet potato (Ipomoea batatas) root tissue was infected by Ceratocystis fimbriata, activity of the enzyme system from mevalonate to isopentenyl pyrophosphate, especially of pyrophosphomevalonate decarboxylase (EC 4.1.1.33), was increased in the noninfected tissue adjacent to the infected region, preceding the furano-terpene production in the infected region. Cutting and incubation of sweet potato slices did not produce furano-terpenes, and only slightly increased the activity of the enzyme system from mevalonate to isopentenyl pyrophosphate. The enzymic activity in diseased tissue was localized in the soluble fraction, and was higher in the tissue from the surface to a depth of about 5 mm with gradual decrease toward the inner part.     

Screening of <i>Bacillus subtilis</i> HAAS01 and Its Biocontrol Effect on <i>Fusarium</i> wilt in Sweet Potato

Fusarium wilt, a disease caused by Fusarium oxysporum f.sp batatas (Fob) is an important disease in sweet potato production. Using endophytic bacteria for biological control of sweet potato diseases is one of the important ways. A Bacillus subtilis with antagonistic effect on Fusarium wilt of sweet potato was isolated from soil by confrontation culture. According to the biological characteristics, 16S rDNA sequence analysis, and physiological and biochemical analysis, the Bacillus subtilis HAAS01 was named. A pot experiment was conducted for the biological control experiment of strain HAAS01, and the endogenous hormone content, antioxidant enzyme activity, soluble protein content, and related gene expressions of sweet potato plants were detected. The results showed that the HAAS01 strain could promote the production of endogenous hormones and resist the infection of plant diseases together with defensive enzymes and upregulation of related gene expressions. In summary, Bacillus subtilis HAAS01 was effective in controlling Fusarium wilt of sweet potato and has potential for application and development.     

Cytological analysis of tetraploid hybrids between sweet potato and diploid Ipomoea trifida (H. B. K.) Don.

Summary Tetraploid F₁ hybrids between Ipomoea batatas, sweet potato (2n = 6x = ca. 90), and diploid (2n = 2x = 30) I. trifida (H. B. K.) Don. showed various degrees of fertility reduction. The present study aimed to clarify its causes by cytological analysis of meiotic chromosome behavior in the diploid and sweet potato parents and their tetraploid hybrids. The diploid parents showed exclusively 15 bivalents, and the sweet potato parents exhibited almost perfect chromosome pairing along with predominant multivalent formation. Their hybrids (2n = 4x= 57–63) formed 2.6–5.0 quadrivalents per cell, supporting the autotetraploid nature. The meiotic aberratios of the hybrids were characterized by the formation of univalents, micronuclei, and abnormal sporads (monad, dyad, triad, and polyad). The causes underlying these aberrations were attributed in part to the multivalent formation, and in part to a disturbance in the spindle function. Three hybrids showing serious meiotic aberrations were very low in fertility. The utilization of the sweet potato-diploid I. trifida hybrids for sweet potato improvement is described and, further, the role of interploidy hybridization in the study of the sweet potato evolution is discussed.     

Molecular genetic analysis of virus isolates from wild and cultivated plants demonstrates that East Africa is a hotspot for the evolution and diversification of Sweet potato feathery mottle virus

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) is globally the most common pathogen of cultivated sweet potatoes (Ipomoea batatas; Convolvulaceae). Although more than 150 SPFMV isolates have been sequence‐characterized from cultivated sweet potatos across the world, little is known about SPFMV isolates from wild hosts and the evolutionary forces shaping SPFMV population structures. In this study, 46 SPFMV isolates from 14 wild species of genera Ipomoea, Hewittia and Lepistemon (barcoded for the matK gene in this study) and 13 isolates from cultivated sweet potatoes were partially sequenced. Wild plants were infected with the EA, C or O strain, or co‐infected with the EA and C strains of SPFMV. In East Africa, SPFMV populations in wild species and sweet potato were genetically undifferentiated, suggesting inter‐host transmission of SPFMV. Globally, spatial diversification of the 178 isolates analysed was observed, strain EA being largely geographically restricted to East Africa. Recombination was frequently detected in the 6K2‐VPg‐NIaPro region of the EA strain, demonstrating a recombination ‘hotspot’. Recombination between strains EA and C was rare, despite their frequent co‐infections in wild plants, suggesting purifying selection against strain EA/C recombinants. Positive selection was predicted on 17 amino acids distributed over the entire coat protein in the globally distributed strain C, as compared to only four amino acids in the coat protein N‐terminus of the EA strain. This selection implies a more recent introduction of the C strain and a higher adaptation of the EA strain to the local ecosystem. Thus, East Africa appears as a hotspot for evolution and diversification of SPFMV.     

Increase in mitochondrial activity in diseased sweet potato root tissue

When infected with Ceratocystis fimbriata, sweet potato roottissue showed an increase in respiration concomitant with anincrement in mitochondrial activities, which was not in parallelwith the increase in mitochondrial numbers. ¹ This paper constitutes part 100 of the phytopathological chemistryof sweet potato with black rot and injury. ² Present address: Department of Biochemistry, Faculty of NaturalSciences, Komensky University, Bratislava, Czechoslovakia. (Received June 21, 1972; )     

Inhibition of the gene expression for granule-bound starch synthase I by RNA interference in sweet potato plants

Granule-bound starch synthase I (GBSSI) is one of the key enzymes catalyzing the formation of amylose, a linear α(1,4)D-glucan polymer, from ADP-glucose. Amylose-free transgenic sweet potato plants were produced by inhibiting sweet potato GBSSI gene expression through RNA interference. The gene construct consisting of an inverted repeat of the first exon separated by intron 1 of GBSSI driven by the CaMV 35S promoter was integrated into the sweet potato genome by Agrobacterium tumefaciens-mediated transformation. In over 70% of the regenerated transgenic plants, the expression of GBSSI was inactivated giving rise to storage roots containing amylopectin but not amylose. Electrophoresis analysis failed to detect the GBSSI protein, suggesting that gene silencing of the GBSSI gene had occurred. These results clearly demonstrate that amylose synthesis is completely inhibited in storage roots of sweet potato plants by the constitutive production of the double-stranded RNA of GBSSI fragments. We conclude that RNA interference is an effective method for inhibiting gene expression in the starch metabolic pathway.     

Mixed Crystals of Threonine and Allothreonine

Study of the soluble proteins of sweet potato (Ipomoea batatas L. cv. Norin 1) roots showed that the major protein had an apparent molecular weight of 25,000, and accounted for 60 ~ 70% of the total soluble protein extracted from fresh tissue. The 25-kDa protein exists in two forms, which can be resolved into two bands by nondenaturing polyacrylamide gel electrophoresis. Immunodiffusion and crossed immunoelectrophoresis showed that these forms are immunologically identical. This protein was identified as the antigenic component A of sweet potato root.¹⁾ It was degraded to proteins of lower molecular weight (9,500 to 20,000) if the tissue was cut or infected by Ceratocystis fimbriata. As almost none of this 25-kDa protein was detected in roots stored for one year at 10 ~ 12°C, it is probably the storage protein of these roots. Another major protein was identified as β-amylase by immunodiffusion and immunoelectrophoresis. The amount of β-amylase did not change appreciably after cutting or infection, but it was present in only trace amounts in the roots stored for one year, Cutting, infection, or storage of root tissue resulted in the production of new isozymes of peroxidase, acid phosphatase, and esterase. Increases in some other proteins in cut and in diseased tissues were detected by gel electrophoresis.     

Cloning and characterization of the Rubisco activase gene from <Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam

A full-length cDNA of Rubisco activase (IBrcaI) was cloned from sweet potato (Ipomoea batatas (L.) Lam) using Rapid-Amplification of cDNA Ends (RACE). IBrcaI contains a 1,347 bp open reading frame encoding a protein of 439 amino acids. The sequence alignment of multiple Rubisco activase genes from sweet potato and other plants showed high homology at two previously described ATP-binding sites. Western blot analysis indicated that there are two Rubisco activase proteins in sweet potato. Expression of IBrcaI was only detected in leaves. In the 14 h light and 10 h dark photoperiods, maximal and minimal IBrcaI mRNA expression levels were detected at 8:00 in the morning and at midnight, respectively.     

A sucrose non‐fermenting‐1‐related protein kinase‐1 gene,IbSnRK1, improves starch content,composition, granule size,degree of crystallinity and gelatinization in transgenic sweet potato

Sucrose non‐fermenting‐1‐related protein kinase‐1 (SnRK1) is an essential energy‐sensing regulator and plays a key role in the global control of carbohydrate metabolism. The SnRK1 gene has been found to increase starch accumulation in several plant species. However, its roles in improving starch quality have not been reported to date. In this study, we found that the IbSnRK1 gene was highly expressed in the storage roots of sweet potato and strongly induced by exogenous sucrose. Its expression followed the circandian rhythm. Its overexpression not only increased starch content, but also decreased proportion of amylose, enlarged granule size and improved degree of crystallinity and gelatinization in transgenic sweet potato, which revealed, for the first time, the important roles of SnRK1 in improving starch quality of plants. The genes involved in starch biosynthesis pathway were systematically up‐regulated, and the content of ADP‐glucose as an important precursor for starch biosynthesis and the activities of key enzymes were significantly increased in transgenic sweet potato. These findings indicate that IbSnRK1 improves starch content and quality through systematical up‐regulation of the genes and the increase in key enzyme activities involved in starch biosynthesis pathway in transgenic sweet potato. This gene has the potential to improve starch content and quality in sweet potato and other plants.     

Production of ethylene by sweet potato roots infected by black rot fungus

Ethylene production by sweet potato roots infected by the blackrot fungus, Ceratocystis fimbriata, increased strikingly afterinfection. The fungus grown on potato extract containing 1%sucrose or steamed sweet potato produced no ethylene. Thus,ethylene was proven to be produced from the host tissue affectedby fungus invasion. The ethylene production seemed to be stimulatedby carbon dioxide. Oxygen was essential for production, butexcess oxygen, probably over 80%, was found to be inhibitory.Apparent fungus growth on sweet potato was reduced under a hightension of oxygen, but this was not a cause of reduced ethyleneproduction in excess oxygen. When tissue plugs of infected sweet potato which were activelyproducing ethylene were sliced into thin discs, ethylene productionwas abolished with the exception that the first 1 mm discs atthe 1st and 2nd day stages produced a significant amount ofethylene. Similarly, plugs which were removed from fungus-invadedparts did not produce an appreciable amount of ethylene. Theproduction of ethylene was observed only by tissue plugs whichconsisted of both fungus invaded and noninvaded parts. Infected sweet potato tissue produced ethylene at a rate comparableto that in apples and may provide a goodsystem for the studyof ethylene biosynthesis. ¹Part 72 of the Phytopathological Chemistry of Sweet Potatowith Black Rot and Injury.     

Overexpression of <i>IbSINA5</i> Increases Cold Tolerance through a CBF SINA-COR Mediated Module in Sweet Potato

Seven in absentia (SINA) family proteins play a central role in plant growth, development and resistance to abiotic stress. However, their biological function in plant response to cold stress is still largely unknown. In this work, a seven in absentia gene IbSINA5 was isolated from sweet potato. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses demonstrated that IbSINA5 was ubiquitously expressed in various tissues and organs of sweet potato, with a predominant expression in fibrous roots, and was remarkably induced by cold, drought and salt stresses. Subcellular localization assays revealed that IbSINA5-GFP fusion protein was mainly localized in cytoplasm and nucleus. Overexpression of IbSINA5 in sweet potato led to dramatically improved resistance to cold stress in transgenic plants, which was associated with the up-regulated expression of IbCOR (cold-regulated) genes, increased proline production, and decreased malondialdehyde (MDA) and H₂O₂ accumulation in the leaves of transgenic plants. Furthermore, transient expression of IbCBF3, a C-repeat binding factor (CBF) gene, in the leaf protoplasts of wild type sweet potato plants up-regulated the expression of both IbSINA5 and IbCOR genes. Our results suggest that IbSINA5 could function as a positive regulator in the cold signaling pathway through a CBF-SINA-COR mediated module in sweet potato, and have a great potential to be used as a candidate gene for the future breeding of new plant species with improved cold resistance.     

Characterization of the wound-inducible protein ipomoelin from sweet potato

The ipomoelin (IPO) gene, a wound- and methyl jasmonate-inducible gene, was isolated from sweet potato (Ipomoea batatas cv. Tainung 57), and previously demonstrated to be regulated by dephosphorylated proteins and calcium ion (Chen Y.-C. et al. Plant Cell and Environment 26, 1373–1383, 2003). In this report, the function of the IPO protein was further studied. The IPO gene was characterized as having one intron and presenting two copies within the genome of sweet potato. The IPO protein appeared 1 d after the leaves of sweet potato were wounded. Surprisingly, the accumulation of the IPO protein remained for 7 d after wounding. Additionally, after the IPO protein was fused to a histidine tag , the His-IPO fusion protein produced from Escherichia coli BL21DE3 was then used to perform the haemagglutination test, which demonstrated that His-IPO fusion protein agglutinated human blood cells. Furthermore, several carbohydrates, including methyl α- d -glucopyranoside, methyl α- d -mannopyranoside, maltose, mannose, glucose, galactose, and lactose, reduced the efficiency of the His-IPO fusion protein in agglutinating human blood cells. These experimental results may indicate that the IPO protein is a lectin , a carbohydrate-binding protein. Notably, the IPO protein retarded the growth and development of silkworm, and thus reduced silkworm survival rates. Therefore, these findings indicate that the function of the IPO protein is to protect plants from insect attack.     

甘薯丛枝病植原体的PCR检测

以报道的植原体 (Phytoplasma)16SrDNA基因保守序列为依据,设计合成了两对引物对R16mF2/ R16mR2和R16F2/ R16R2,以甘薯丛枝病（SPWB）带病植株的叶脉中提取的DNA为模板,应用聚合酶链式反应（PCR）技术和巢式PCR（Nested- PCR）技术对甘薯丛枝病病原进行分子检测。结果表明PCR扩增出了1.5　kb的特异片段,在PCR基础上的巢式PCR扩增出了1.2　kb的特异片段。灵敏度实验显示该方法所需PCR模板DNA量为0.1073　ng/μl,在PCR基础上的巢式PCR可以将灵敏度提高约10000倍,所需模板DNA仅为0.01073　pg/μl,在甘薯丛枝病的检测中是一种快速、灵敏、可靠的方法。     

基于农业废弃物培育优质蛹虫草的研究

利用农业废弃物甘薯藤及蛹虫草培养基废弃物作为培养基的主要原料进行蛹虫草菌种驯化。蛹虫草子实体培养基中添加不同比例蛹虫草培养基废弃物及甘薯藤粒,通过适宜的培养条件,废弃物中淀粉、蛋白质、糖类、氨基酸等营养物质及蛹虫草胞外酶酶解产生的小分子物质被充分利用,以培育优质蛹虫草子实体。当一级种子中加入蛹虫草培养基废弃物20 g/L和甘薯藤粉10 g/L,二级种中加入蛹虫草培养基废弃物20 g/L、甘薯藤粉10 g/L,使蛹虫草子实体栽培料中蛹虫草培养基废弃物占32%～45%,甘薯藤粒占10%～15%,二者比例为(3～4)：1时,栽培效果最好。本研究蛹虫草培养基替代原料资源丰富易得,并可节约生产用粮,降低原料成本,从而实现农用废弃物再利用,减少环境污染,也符合绿色环保可持续发展的理念。     

甘薯丛枝病植原体的PCR检测

以报道的植原体（Phytoplasma)16SrDNA基因保守序列为依据,设计合成了两对引物对R16mF2/R16mR2和R16F2／R16R2,以甘薯丛枝病（SPWB）带病植株的叶脉中提取的DNA为模板,应用聚合酶链式反应（PCR）技术和巢式PCR（Nested-PCR)技术对甘薯丛枝病病原进行分子检测。结果表明PCR扩增出了1．5kb的特异片段,在PCB基础上的巢式PCR扩增出了1．2kb的特异片段,灵敏度实验显示该方法所需PCR模板DNA量为0．1073ng/ul在PCR的基础上的巢度PCR可以将灵敏度提高约10000倍,所需模板DNA仅为0．01073pg/ul,在甘薯丛枝病的检测中是一种快速,灵敏,可靠的方法。