Occurrence and survival of potato scab pathogens (Streptomyces species) on tuber lesions: quick diagnosis based on a PCR-based assay

A time-saving and cost-effective polymerase chain reaction (PCR)-based method was developed for species-specific detection of the scab pathogens ( Streptomyces scabies and S. turgidiscabies ) prevalent in potato ( Solanum tuberosum ) in northern Scandinavia. Species specificity of primers was verified using a collection of previously characterized Streptomyces strains isolated from potato scab lesions in Finland and Sweden. A total of 1245 scab lesions was tested from potato cvs Matilda and Sabina grown in the field in two geographic regions of Finland in 2000 and 2001. Freshly harvested or stored potato tubers were incubated at room temperature (18–21°C) under humid conditions for a few days. Bacterial growth was collected from scab lesions for DNA isolation and PCR. The two scab pathogens were detected in the same potato fields, tubers and scab lesions. The relative incidence of S. scabies was high in freshly harvested tubers but was much lower than that of S. turgidiscabies following storage. Both pathogens were seed-transmitted in Matilda and Sabina after 24 weeks of storage at 4°C.     

Differential effect of nematicide treatments on tuber yields in early- and late-maturing potato cultivars

A 3-year field study examined the effects of 1,3-dichloropropene or oxamyl on tuber yields in four early- and five late-maturing potato cultivars. The nematicide treatments increased total tuber yield by ≈ 12% for early-maturing cultivars, but by less than 2% for the late-maturing cultivars. The treatments reduced the numbers of root lesion nematodes (primarily Pratylenchus penetrans ), which were the dominant plant parasitic nematodes at the site. Covariance analysis indicated that treatment effects on total tuber yield were no longer significant after adjusting for root lesion nematodes in soil at harvest, confirming that these nematodes had a direct effect on the host. As expected, there were significant inverse relationships ( P  = 0·05) between total tuber yields and numbers of root lesion nematodes in soil and in roots. However, the application of nematicides in late-maturing cultivars was found to be of no practical value. Crop production systems designed to reduce or optimize pesticide applications can form one criterion in the development of more environmentally sensitive management strategies. The merit of utilizing chemicals for root lesion nematode control are considered with regard to cultivar maturity.     

干旱与复水对马铃薯块茎膨大期碳氮转运的影响

为探讨干旱及复水对马铃薯块茎膨大期碳氮物质转运影响的生理机制,采用盆栽控水法研究了干旱胁迫、旱后复水过程中马铃薯叶片与块茎中碳水化合物、氮素同化物以及碳氮转运相关酶活性的变化规律。结果表明：干旱胁迫下马铃薯叶片蔗糖磷酸合成酶（SPS）活性显著增强,增幅为54.9%,可溶性糖与蔗糖含量显著增加,分别增加84%、25%;块茎蔗糖与淀粉含量显著下降,分别降低25%、11.8%。复水后叶片可溶性糖含量变化较小,叶片SPS活性仍保持较高水平;块茎中蔗糖合成酶（SS）活性下降6.25%(P＜0.05),蔗糖与淀粉含量分别增加21.8%、29.8%。干旱胁迫下马铃薯叶片中硝酸还原酶（NR）活性下降49.3%,谷氨酰胺合成酶（GS）活性与对照差异不显著,叶片游离氨基酸与可溶性蛋白质含量分别降低29.4%、38.4%,全氮含量降低13%;块茎中游离氨基酸与可溶性蛋白质含量增加16.8%、270.9%。复水后叶片中NR与GS活性高出对照3.5%、12.1%,叶片游离氨基酸、可溶性蛋白质以及全氮含量仍保持较低水平;块茎游离氨基酸、可溶性蛋白含量较高,增幅分别为24.6%、178.4%,全氮含量增加,与对照差异不显著。说明干旱胁迫显著阻碍了马铃薯碳水化合物与氮素同化物的合成与转运,使马铃薯叶片碳水化合物积累,但干旱促进了氮素向块茎转运,块茎中的碳氮比下降;复水后马铃薯植株能够快速修复干旱胁迫造成的损伤,表现为块茎中淀粉含量增多,块茎碳氮比增加。     

Root infection of potato by Spongospora subterranea: knowledge review and evidence for decreased plant productivity

Information is reviewed on root infection of potato by the plasmodiophorid Spongospora subterranea f. sp. subterranea. This pathogen has long been recognized as the cause of root galls (hyperplasia) and the economically important disease powdery scab on tubers (modified stolons). The significance for plant productivity of the zoosporangium stages of the pathogen in potato roots has only recently begun to be documented. Two experiments are described that assessed effects of S. subterranea root infection on potato plant root function and productivity. A greenhouse experiment measured root function and plant parameters for eight potato cultivars with markedly different susceptibilities to tuber powdery scab. Water uptake and plant growth were reduced by S. subterranea inoculation in all eight cultivars. The magnitudes of these negative effects, and intensities of root hyperplasia, differed among the cultivars, but were not related to respective susceptibilities to tuber powdery scab. A field trial assessed root function and plant productivity for a cultivar (Iwa) that is very susceptible to Spongospora tuber and root diseases. Soil water content beneath uninoculated plants was consistently less than for inoculated plants, indicating that inoculation reduced water uptake (root function). Inoculation reduced shoot and root dry weights, and reduced weight of tubers per plant by 42%. Spongospora subterranea causes three diseases of potato: root membrane dysfunction, root hyperplasia and tuber powdery scab. The root diseases caused by the pathogen are likely to be important both for powdery scab management and for deleterious effects on potato crop yields.     

甘肃省马铃薯镰刀菌干腐病优势病原的分离鉴定

 为明确甘肃马铃薯镰刀菌干腐病的优势病原,2006 年12 月~ 2007 年3 月由西至东从甘肃张掖、天祝、永登、临洮、渭源和西和等6 县市的马铃薯贮藏窖中采集表现有镰刀菌干腐病症状的马铃薯薯块,以组织分离法分离病原,单孢纯化镰刀菌(Fusarium spp. )菌株后,以形态学为基础,参照Nelson 镰刀菌分类系统进行鉴定。结果表明:6 个采样区共分离到293株镰刀菌菌株,其中以接骨木镰刀菌(F. sambucinum)和茄病镰刀菌(F. solani)出现频率高,是优势种。分析发现第一优势种随采样区而异,张掖、天祝和渭源采集的样品中茄病镰刀菌分离频率分别为42. 6% 、42. 1% 和32. 4% ,接骨木镰刀菌分离频率分别为14. 8% 、5. 3% 和26. 5% ,茄病镰刀菌为第一优势种;永登、临洮和西和采集的样品中接骨木镰刀菌分离频率分别为52郾1% 、50. 9% 和55. 2% ,茄病镰刀菌的分离频率分别为23. 3% 、32. 7% 和20. 7% ,接骨木镰刀菌为第一优势种。本文进一步对其在PDA、CLA 上的培养特征进行了观察和描述。按照柯赫氏法则用混合菌株接种法对大西洋(Atlantic)、夏波蒂(Shepody)以及一地方品种进行致病性测定,证实了优势菌种对块茎的致病性。利用EF鄄1琢基因引物(EF鄄1H 和EF鄄2T)对接骨木镰刀菌菌株GAUF鄄F12 进行基因组DNA 的PCR 扩增,将PCR 产物回收测序后在GenBank 上比对,菌株GAUF鄄F12 与GenBank 登记的接骨木镰刀菌5 个菌株的同源性均达99% ;用DNASTAR 分析软件将同源性较高的登记菌株的序列与GAUF鄄F12 菌株构建同源性树,结果表明:该菌株与以上5 个接骨木镰刀菌菌株均位于同源性树的同一分支,聚为一类,与形态学的鉴定结果一致。     

不同探针大小、标记物及反应底物对马铃薯类病毒杂交检测的影响

 灵敏可靠地检测马铃薯纺锤块茎类病毒(Potato spindle tuber viroid,PSTVd)对脱毒种薯的生产具有重要意义。本研究探索了影响核酸杂交检测技术的关键因素。通过基因克隆技术构建了插有PSTVd全长单体、双体及片段的载体。分别以地高辛和同位素为标记物,利用PCR和转录标记技术制备cDNA和RNA探针。比较探针大小、标记物、标记方法、反应底物等对检测灵敏度的影响。结果显示,以地高辛为标记物,利用PCR标记制备的PSTVd双体cDNA探针,在以CDP-Star为底物,通过在柯达X-OMAT BT胶片进行化学发光反应来分析结果的检测灵敏度最高,可以检测到0.05 pg总RNA中的PSTVd,是国外报道检测灵敏度的500倍。利用核酸斑点杂交技术检测PSTVd具有灵敏度高,一次可检测样品数量多等特点,对于大规模PSTVd检测更加方便可行。     

不同复种油菜-轮作模式对马铃薯耗水特征及产量的影响

为探讨马铃薯不同轮作系统复种饲用油菜对马铃薯耗水特征及产量的影响,2017年在长期大田定位试验基础上选取4个处理,即马铃薯连作（CK）、马铃薯-小麦复种油菜-马铃薯（WPRP）、马铃薯-豌豆复种油菜-马铃薯（PPRP）、小麦-豌豆复种油菜-马铃薯（WPPR）,研究不同复种轮作模式对马铃薯耗水特性、水分利用效率和产量的影响。结果表明：马铃薯块茎增长期到收获期,CK 160~200 cm土壤水分含量和播前无差异,而WPRP、PPRP、WPPR 0~200 cm土壤各层含水量均大于播前,其中,块茎增长期到淀粉积累期,各层土壤平均含水量分别提高了43.02%、38.58%、24.63%,淀粉积累期到收获期,分别提高36.47%、33.63%和21.43%; 120~200 cm土壤各层含水量表现为：WPRP＞PPRP＞WPPR＞CK。WPRP、PPRP、WPPR耗水深度和贮水量均大于CK,其中,块茎增长期到淀粉积累期,耗水深度较CK分别增加42.86%、42.86%、42.86%,土壤贮水量较CK分别增加10.36%、12.91%和8.49%;淀粉积累期到收获期,耗水深度较CK分别增加了25.00%、12.50%、12.50%,土壤贮水量分别增加了12.56%、6.78%和2.95%,且WPRP贮水量较CK显著提高12.56%。块茎形成期、块茎增长期和淀粉积累期,WPRP耗水量较CK分别减少了16.2%、25.2%和5.1%。WPRP、PPRP、WPPR较CK产量分别增加33.51%、40.05%、14.47%,WUE分别提高54.83%、52.32%、19.78%。综上,马铃薯轮作系统复种油菜增加了降雨入渗深度、土壤贮水量和耗水深度,降低了阶段性耗水量,提高了马铃薯产量和WUE。且马铃薯-小麦复种油菜-马铃薯为该地区较佳的轮作模式。     

Histopathology of infection and colonization of Quercus ilex fine roots by Phytophthora cinnamomi

Quercus ilex is one of the European forest species most susceptible to root rot caused by the oomycete Phytophthora cinnamomi. This disease contributes to holm oak decline, a particularly serious problem in the ‘dehesas’ ecosystem of the southwestern Iberian Peninsula. This work describes the host–pathogen interaction of Q. ilex and P. cinnamomi, using new infection indices at the tissue level. Fine roots of 6‐month‐old saplings inoculated with P. cinnamomi were examined by light microscopy and a random pool of images was analysed in order to calculate different indices based on the measured area of pathogen structures. In the early stages of invasion, P. cinnamomi colonizes the apoplast and penetrates cortical cells with somatic structures. On reaching the parenchymatous tissues of the central cylinder, the pathogen develops different reproductive and survival structures inside the cells and then expands through the vascular system of the root. Some host responses were identified, such as cell wall thickening, accumulation of phenolic compounds in the middle lamella of sclerenchyma tissues, and mucilage secretion blocking vascular cells. New insights into the behaviour of P. cinnamomi inside fine roots are described. Host responses fail due to rapid expansion of the pathogen and a change in its behaviour from biotrophic to necrotrophic.     

Differences in the responses of melon accessions to fusarium root and stem rot and their colonization by Fusarium oxysporum f. sp. radicis‐cucumerinum

Fusarium root and stem rot caused by the fungus Fusarium oxysporum f. sp. radicis‐cucumerinum is a major disease in greenhouse cucumbers. Over the past decade, the disease has been documented in melon greenhouses in Greece, and recently it has been sporadically recorded in greenhouse melons in Israel. Variations in disease response were found among 41 melon accessions artificially inoculated with the pathogen: 10 accessions were highly susceptible (90–100% mortality), 23 exhibited an intermediate response (20–86%) and eight were resistant (0–4%). Two melon accessions – HEM (highly resistant) and TAD (partially resistant) – were crossed with the susceptible accession DUL. The responses of the three accessions and F₁ crosses between the resistant and susceptible parents were evaluated. HEM contributed higher resistance to the F₁ hybrid than TAD. Roots of susceptible and resistant accessions were 100 and 79% colonized, respectively, following artificial inoculation. However, only susceptible plants showed colonization of the upper plant tissues. Microscopic evaluation of cross sections taken from the crown region of the susceptible DUL revealed profuse fungal growth in the intercellular spaces of the parenchyma and in xylem vessels. In the resistant cultivar HEM, very little fungal growth was detected in the intercellular spaces of the parenchyma, and none in the xylem or any other vascular tissue. Finding resistant accessions may create an opportunity to study the genetics of resistance inheritance and to develop molecular markers that will facilitate breeding resistant melon cultivars.     

Effect of soil inoculum level and environmental factors on potato powdery scab caused by Spongospora subterranea

The effects of soil inoculum level and three environmental factors (soil type, soil moisture regime and temperature) on the incidence and severity of powdery scab caused by Spongospora subterranea were investigated in potato plants grown under controlled environmental conditions. Symptoms of powdery scab on tubers were assessed visually, after which DNA was extracted from tuber peelings and quantified in a real-time polymerase chain reaction assay using primers and a TaqMan® probe specific to S. subterranea to establish tuber infection levels. Soil inoculum concentration of S. subterranea did not significantly affect the incidence and severity of either tuber infection or powdery scab symptoms at maturity. No significant differences in disease incidence and severity were found between sandy, loamy and clay soils, although the two lighter soils yielded more powdery scab than clay soil. Constant dampness of the soil resulted in significantly more disease than a fluctuating moisture regime. Infection and disease levels were high at all three temperatures tested (9, 12 and 17°C), but symptoms were most severe at 12°C. The percentage of plants with infected tubers did not increase after tuber initiation, although the amount of S. subterranea DNA detected in tubers and the severity of powdery scab symptoms increased in mature plants. Latent tuber infections were found to be common, especially under conditions suboptimal for disease development. This new information may be important for the prevention of powdery scab in potato-growing areas around the world.     

河北省甘薯镰孢菌腐烂与溃疡病的病原鉴定

为明确河北省甘薯镰孢菌腐烂与溃疡病的症状特点和病原种类,在不同种植区采集储藏期和育秧期病样,描述其症状特征;对病原菌进行分离纯化,采用柯赫氏法则回接验证,依据病原菌的形态特征和基因序列确定病原菌种类。结果表明,在储藏期甘薯发病可导致薯块表面腐烂的占总病薯的59.09%,端部腐烂的占40.91%;薯块带有褐色轮纹病斑的占61.36%,病斑没有轮纹或者轮纹不明显的占38.64%;发病初期薯块内部病斑浅、黑褐色的占27.27%,发病后期薯块内部形成空腔、布满白色菌丝的占72.73%;病斑带有苦味的占59.09%,病斑没有苦味或苦味不明显的占40.91%;在育秧田发病导致薯秧溃疡,表现为主茎基部呈点片发生黑色或者褐色病斑,部分有开裂。分离的病原菌能够同时侵染薯块和薯秧;病原菌单瓶梗产孢,大型分生孢子稍弯,两端钝圆,多数3~7个分隔,顶细胞钝圆,基细胞足跟较明显。其rDNA-ITS、EF-1α、β-tubulin基因序列与茄镰孢菌Fusarium solani的同源性分别为97%、99%和98%。初步确定甘薯镰孢菌腐烂与溃疡病的病原菌为茄镰孢菌F.solani。     

Control of blackleg and tuber soft rot of potato caused by Pectobacterium and Dickeya species: a review

This paper briefly reviews research on the causative agents of blackleg and soft rot diseases of potato, namely Pectobacterium and Dickeya species, and the disease syndrome, including epidemiological and aetiological aspects. It critically evaluates control methods used in practice based on the avoidance of the contamination of plants, in particular the use of seed testing programmes and the application of hygienic procedures during crop production. It considers the perspective of breeding and genetic modification to introduce resistance. It also evaluates the application of physical and chemical tuber treatments to reduce inoculum load and examines the possibility of biocontrol using antagonistic bacteria and bacteriophages.     

Limited asymptomatic colonization of apple tree shoots by Neonectria ditissima following infection of leaf scars and pruning wounds

European apple canker, caused by Neonectria ditissima, is an important disease of apple (Malus domestica). The fungus may reside in the tree without causing symptoms for up to a few years, thus making canker control difficult. Asymptomatic infections established in the nursery can result in severe canker outbreaks in newly established apple orchards. It has been suggested that N. ditissima might colonize the tree beyond the infection point during the asymptomatic stage. We investigated whether N. ditissima can colonize the internal tissues of apple shoots, both prior to and after visual symptoms. Apple trees were artificially inoculated via pruning wounds and leaf scars; then the pathogen was tracked at the inoculation point and beyond with isolation or real-time quantitative PCR (qPCR). Before visual symptoms, N. ditissima could be detected in the infected pruning cut or leaf scar, but not at a distance of 10–15 mm from the entry point, or greater. Conversely, after symptom expression, the pathogen could be detected in the symptomless tissue at 10–15 mm from a canker lesion. This study demonstrated that the asymptomatic infection by N. ditissima can be detected using qPCR and that the pathogen does not grow systemically much beyond the initial entry point inside the plant before visual canker symptoms appear.     

甘薯茎腐病的研究进展

甘薯茎腐病是由达旦提狄克氏菌Dickeya dadantii Samson et al.引起的细菌性甘薯病害,属于我国新发生病害,其蔓延迅速、防治困难,严重影响甘薯的品质和产量,已成为我国南方甘薯产区主要病害之一。文章对该病症状、病原菌分类、致病机制、全基因组测序概况和防治措施等方面的研究进展进行了综述,并指出了今后的研究方向。     

Biological control of potato soft rot caused by Dickeya solani and the survival of bacterial antagonists under cold storage conditions

Dickeya and Pectobacterium are responsible for causing blackleg of plants and soft rot of tubers in storage and in the field, giving rise to losses in seed potato production. In an attempt to improve potato health, biocontrol activity of known and putative antagonists was screened using in vitro and in planta assays, followed by analysis of their persistence at various storage temperatures. Most antagonists had low survival on potato tuber surfaces at 4 °C. The population dynamics of the best low-temperature tolerant strain and also the most efficient antagonist, Serratia plymuthica A30, along with Dickeya solani as target pathogen, was studied with TaqMan real-time PCR throughout the storage period. Tubers of three potato cultivars were treated in the autumn with the antagonist and then inoculated with D. solani. Although the cell densities of both strains decreased during the storage period in inoculated tubers, the pathogen population was always lower in the presence of the antagonist. The treated tubers were planted in the field the following growing season to evaluate the efficiency of the bacterial antagonist for controlling disease incidence. The potato endophyte S. plymuthica A30 protected potato plants by reducing blackleg development on average by 58.5% and transmission to tuber progeny as latent infection by 47–75%. These results suggest that treatment of potato tubers with biocontrol agents after harvest can reduce the severity of soft rot disease during storage and affect the transmission of soft rot bacteria from mother tubers to progeny tubers during field cultivation.     

Trichothecene production is detrimental to early root colonization by Fusarium culmorum and F. graminearum in fusarium crown and root rot of wheat

Fusarium culmorum (Fc) and F. graminearum (Fg) belong to the predominant causal agents of fusarium crown and root rot (FCR) in wheat. While many studies have been done to investigate crown rot, including stem base infection, root colonization and mycotoxin production associated with root rot is not well understood. In this study the impact of mycotoxins on the colonization of wheat roots and stem bases was analysed by using Fc and Fg isolates that varied in both quantity and types of trichothecenes they produce. Seedling inoculations in growth chambers with a high deoxynivalenol (DON)- and 3-acetyldeoxynivalenol (3ADON)-producing isolate led to more severe symptoms and 20-times greater colonization of the stem base, as measured by Fc DNA accumulation, than isolates that produced less DON/3ADON. In contrast to stem base colonization, in vitro inoculations of roots with a Tri5 deletion mutant deficient in Fg trichothecene production led to three-times higher colonization than the wildtype. Furthermore, an Fc isolate that produced low levels of zearalenone resulted in twice the level of colonization of a high DON/3ADON-producing isolate included in the study. When root inoculation with a low DON/3ADON-producing Fc isolate was supplemented with exogenous DON, DON production decreased by more than half per unit weight of Fc DNA, and root colonization doubled compared to the untreated control. Therefore, in contrast to its potential role as an aggressiveness factor in stem base infection, trichothecene production by Fc and Fg is detrimental to the early stages of wheat root colonization in FCR.     

温湿度调控对番茄灰霉病菌产生的细胞壁降解酶的影响

 番茄灰霉病菌在致病过程中能够产生4种细胞壁降解酶,以PMG酶活性最高,其次是β-葡萄糖苷酶和PG酶,Cx最少。灰霉病菌在不同温度下侵染番茄叶片时产生的致病酶活性不同,4种酶在20℃时表现了最高的活性,15℃次之,当温度达到25℃时,各种酶的活性都急剧下降,随着温度的再升高,酶活更低。随着湿度的增高,病菌产生的细胞壁降解酶的活性也增加,当相对湿度达到90%以上时,4种酶的活性也达到最高。温湿度对番茄灰霉病菌产生细胞壁降解酶的影响趋势,与其对发病的影响趋势是一致的。     

Role of co‐infection by Pectobacterium and Verticillium dahliae in the development of early dying and aerial stem rot of Russet Burbank potato

Potato early dying (PED) is a disease complex primarily caused by the fungus Verticillium dahliae. Pectolytic bacteria in the genus Pectobacterium can also cause PED symptoms as well as aerial stem rot (ASR) of potato. Both pathogens can be present in potato production settings, but it is not entirely clear if additive or synergistic interactions occur during co‐infection of potato. The objective of this study was to determine if co‐infection by V. dahliae and Pectobacterium results in greater PED or ASR severity using a greenhouse assay and quantitative real‐time PCR to quantify pathogen levels in planta. PED symptoms caused by Pectobacterium carotovorum subsp. carotovorum isolate Ec101 or V. dahliae isolate 653 alone included wilt, chlorosis and senescence and were nearly indistinguishable. Pectobacterium wasabiae isolate PwO405 caused ASR symptoms including water‐soaked lesions and necrosis. Greater Pectobacterium levels were detected in plants inoculated with PwO405 compared to Ec101, suggesting that ASR can result in high Pectobacterium populations in potato stems. Significant additive or synergistic effects were not observed following co‐inoculation with these strains of V. dahliae and Pectobacterium. However, infection coefficients of V. dahliae and Ec101 were higher and premature senescence was greater in plants co‐inoculated with both pathogens compared to either pathogen alone in both trials, and V. dahliae levels were greater in basal stems of plants co‐inoculated with either Pectobacterium isolate. Overall, these results indicate that although co‐infection by Pectobacterium and V. dahliae does not always result in significant additive or synergistic interactions in potato, co‐infection can increase PED severity.     

Development of a novel inducible bioluminescent and antibiotic resistance tagging system and its use to investigate the role of antibiotic production by <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> ssp. <Emphasis Type="Italic">carotovorum</Emphasis> during potato tuber infection

We report the construction of a novel Tn7 vector for the tagging and enumeration of target bacteria from complex microbial communities. The system utilises a cassette for inducible bioluminescence and tetracycline resistance that integrates at a defined neutral position present in most Gram-negative species. We used this approach to chromosomally tag Pectobacterium such that it could be enumerated in mixed consortia without placing a significant bioburden on the tagged strain. Two Pectobacterium strains, a carbapenem antibiotic producer and an isogenic knock-out strain were tagged using this system. The modified Pectobacterium strains were used to compare the extent to which potato tuber-associated and endophytic bacteria can gain advantage and multiply in planta, utilising the nutrients released by a Pectobacterium infection, when the infecting Pectobacterium is either an antibiotic producer (Car+) or a carbapenem knock-out (Car−) strain. We show that the ability to synthesise carbapenem has a significant effect upon Pectobacterium numbers throughout the course of the infection. Whilst limiting the number of other bacterial species, carbepenem production allows the Pectobacterium to replicate to higher titres in the rotting tuber. We anticipate that the Tn7 tagging vector will be of use to other researchers studying ecological interactions in complex environments.