Analysis of Population Genetic Change to Single Oospore Strains of Phytophthora infestans

The occurrence of sexual reproduction accelerates the population genetic variation of Phytophthora infestans and makes it more difficult to control.The systematic analysis of the differentiation of phenotype(mating type and metalaxyl sensitivity)and genotype(mtDNA haplotype and SSR genotype)of 65 single oospore strains of P.infestans was carried out in this article.Five test strains were isolated from Heilongjiang Province and Mongolia Autonomous Region.The experiment results showed that the isolation ratio of metalaxyl resistance(MR:HR)of single oospore strains produced through the cross of medium resistance and high resistant parents was 18:13;the isolation ratio of the metalaxyl resistance(S:MR:HR)of single oospore strains produced through the cross of sensitive and high resistant parents was 4:12:7.The progenies of single oospore strains produced through self-fertility parents with medium resistance were all of the medium resistance.The mating types A1:A2 was greater than 1:1 in single oospore strains of the progenies,which did not conform to the Mendel's law of inheritance.All single oospore strains of the progenies inherited mitochondrial DNA fragments from only one parent.Sexual recombination of single oospore strains was verified by using two pairs of SSR primers(Pi4B and Pi4G).At the locus of Pi4B and Pi4G in the cross of KS-37 and KS-25,the separation frequencies of allele were 19:12 and 14:17,respectively.They produced two new genotype strains.This study could provide a basis for formulating disease control strategies.

Cloning of Potato POTHR-1 Gene and Its Expression in Response to Infection by Phytophthora infestans and Other Abiotic Stimuli

A complete cDNA of potato Phytophthora infestans-induced hypersensitive response-related protein gene (POTHR-1) was cloned using rapid amplification of cDNA ends (RACE) strategy according to a fragment sequence which we had cloned using suppression subtractive hybridization (SSH) technique. The potato POTHR-1 gene encodes a protein of 225 amino acids, which shares 81% identity with tobacco hin1 gene-enoded protein (harpin-induced protein). Southern blot revealed that there are two to three copies of POTHR-1 in potato genome. The POTHR-1 gene expression in potato leaves showed that its transcripts accumulated remarkably in leaves after 36 h inoculation with P. infestans. Mechanical wounding and jasmonic acid (JA) could induce the POTHR-1 gene expression and osmotic stress just induce a slight accumulation of POTHR-1 gene mRNA, while salicylic acid (SA) had no detectable function on the induction accumulation of POTHR-1 gene transcripts. The potato POTHR-1 gene may preferentially associate with hypersensitive response (HR) or biotic cell death during interaction between host and pathogen.

Exogenously applied methyl Jasmonate induces early defense related genes in response to Phytophthora infestans infection in potato plants

In order to elucidate the contributions of JA in orchestrating disease resistance in potato plants,the potato genotype‘SD20’,which exhibits strong resistance against the highly virulent Phytophthora infestans isolate CN152,while infected by the super virulent isolate 2013-18-306,was treated with exogenous JA and then challenged by inoculation with 2013-18-306.The results showed that exogenously applied JA significantly delayed the onset and alleviated the symptoms of late blight,indicating exogenous JA could induce resistance to P.infestans in the early biotrophic stage of infection in‘SD20’plants.To further clarify the role of JA in the early defense response and identify key genes involved in JA signal transduction,gene expression profiling via RNA sequencing(RNA-seq)in‘SD20’plants treated with exogenously applied JA was performed.A total of 2927 differentially expressed genes were specifically induced,the majority encoded transcription factors,protein kinases,secondary metabolites,defense enzymes and disease resistance related proteins.GO functional annotation and KEGG metabolic pathway analysis showed that exogenously applied JA rapidly induced the expression of genes related to immune response regulation,pathogen defense,and other biological processes,and stimulated endogenous JA synthesis and signal transduction,and the overall early pathogen defense response in‘SD20’.These results provide useful information in understanding the JA’s function involved in pathogen defense responses and a theoretical basis for the application of JA in potato production.

Potato late blight caused by Phytophthora infestans:From molecular interactions to integrated management strategies

Over 170 years after the infamous Irish Potato Famine,potato late blight(PLB)caused by Phytophthora infestans remains the single most devastating disease of global potato production,causing up to 10 billion USD in yield loss and management costs.Through decades of research,growers and agronomists in the field as well as laboratory scientists have made significant progress in understanding the molecular pathogenesis process of this critical pathosystem and effective management strategies to control PLB.Yet,the need to feed an ever-increasing global population under changing climate demands continued improvement in efficient and sustainable PLB management schemes that can be implemented across a broad economic spectrum.In this review,we briefly summarize the current understanding of the molecular interaction between P.infestans and its host plants,highlight the current integrated pest management strategy to control PLB on local and continental scales,and discuss the potential of further improvement of sustainable PLB control through genetic enhancement of crop resistance and emerging crop protection technologies.

Sensitivities of Phytophthora infestans to Metalaxyl, Cymoxanil, and Dimethomorph

The isolates of Phytophthora infestans on tomato in the Guangxi Zhuang Autonomous Region, China, were determined for the sensitivities to metalaxyl, cymoxanil and dimethomorph to give the basic information for integrating disease management. Sensitivities were tested by measuring the radial growth on agar medium amended with fungicide, compared with the floating-leaf-disk method. 239 isolates were collected from eight tomato growing areas during 2000-2006. The testing results indicated that the frequencies of sensitive, intermediate, and resistant isolates to metalaxyl were 42.26, 35.98, and 23.53%, respectively. Variations in sensitivities amongst isolates from different areas or different years were very high for metalaxyl. All isolates from Tianlin and Wuxuan were sensitive to metalaxyl, but the metalaxyl-resistant isolates predominated in Tianyang, with the frequency of 51.35%. The EC50 values of certain isolates from Tianyang were higher than 500 μg·mL^-1 and their resistance levels were over 100 000 folds. Cymoxanil has been used for nearly 10 years in Guangxi, and dimethomorph has been used for 5-6 years. However, there was no decrease in sensitivity of P. infestans populations and the sensitivities of the pathogen were nearly normally distributed. Hence, their mean ECs0 value [cymoxanil （0.1647 ±0.0255） μg·mL^-1, dimethomorph （0.0970 ± 0.0052） μg·mL^-1 could be used as the baseline sensitivities for monitoring the field resistance development. The comparison with the floating-leaf-disk method indicates that both the techniques provided equivalent results. These studies suggested that metalaxyl can be continuously applied in Tianlin, Wuxuan, and Nanning due to the resistant isolates that have not been found, while for those areas with resistant isolate, the use of metalaxyl should be reduced or alternated, and cymoxanil or dimethomorph was recommended for controlling late blight disease of tomato.

Race Structure and Distribution of Phytophthora infestans in the Investigated Areas of China

Ninety isolates collected from five different provinces and cities (Hebei, Yunan, Sichuan, Chongqing, Inner Mongolia ) during 1997 - 1998 were tested with potato clones possessed 8 dominant mono-genes R1, R3, R4, R6, R7, R9, R10, R11 and the combination of R1-R4 of the international late blight differential host. It concluded that: (1) in the 90 isolates tested 21 different races were detected, of which the 1.3.4. 7. 9. 10. 11 was very common with frequency of 32. 2%, followed by 1. 3. 4. 6. 7. 9. 10. 11 and 3.4.7. 9. 11 with frequency of 10.0%; (2) all tested resistant genes were compatible to the virulent gene of isolates of Phytophthora infestans assayed, which indicated that the virulent genes were very common in the tested population of Phytophthora infestans.

Study on the Relationship Betweenthe Toxin of Phytophthora infestans(Mont.) de Bary and Resistance of Potato

Plant pathogenic fungi produce mycotoxins which may lead host plants to produce disease symptoms and may have a significant positive role in the course of disease development.The putative Phytophthora infestans toxins were prepared by culturing in liquid medium（60 g rye and 10% tomato juice L-1） for 1 mon at 17℃,filtering through four layers of cheesecloth and being precipitated by ammonium sulfate.The resulted putative toxin solutions were used to study the effects on 3 potato varieties（both leaf and tuber tissues）.The results show that potato leaves and tubers exhibit symptoms similar to the late blight resulted from P.infestans infection.Potato varieties reacting differentially to both the toxin dilutions and toxins produced by different P.infestans isolates suggested the presence of toxin-mediated specificities between potato and P.infestans.Potato leaf and tuber tissues have different and contrary reactions to the toxin.Toxin-mediated specificities are likely present between potato and P.infestans.

A Preliminary Investigation on Phenotypic Characteristics of Phytophthora infestans from Guizhou Province

Abstract [ Objective ] This study aimed to investigate the phenotypic characteristics of Phytophthora infestans from Guizhou Province. The metalaxyl resistance, mating type and isozyme genotype of 292 P. infestans strains isolated from Guizhou Province were analyzed. [ Result] Metalaxyl-resistant P. infestans strains were found in different areas of Guizhou Province. Resistant, moderately resistant and susceptible strains accounted for 35.62% , 33.90% and 30.48% , re- spectively. Both mating types A1 and A2 were found, which accounted for 65.38% and 34.62%, respectively. P. infestans strains isolated from Guizhou Province mainly belonged to Gpi genotypes 100/100 and 100/100/111, accounting for 50.68% and 43.84%, respectively. [ Conclusion] This study provides the theoretical basis for the prevention and control of potato late blight in Guizhou Province.

Optimization of PCR System in EST-SSR Analysis of Phytophthora infestans

Late blight caused by the oomycete Phytophthora infestans Montagne de Bary is a devastating disease on potato production. A total of six parameters affecting the PCR system in P. infestans were investigated based on the template DNA of the isolate HH06-23 and EST-SSR primer pair Pi08N. The results showed that the optimal annealing temperature was 63 ℃, and the optimum PCR system of EST-SSR was 25 ng template DNA, 0.5 mmol·L^-1 dNTPs, 2 μL 10×Buffer （Mg^2＋ free）, 1.75 mmol·L^-1 MgCl2, 15 pmol primer, and 1.2 U Taq DNA polymerase in total 20 μL reaction system. The PCR program was initial denaturation at 94℃ for 2 rain, followed by 35 cycles of 94℃ for 30 s, 63℃ for 30 s, and 72℃ for 30 s, then a final extension step was 72℃ for 7 min, and held at 4℃. In addition, using the optimal PCR system, a total of 20 isolates of P. infestans were used for testing the stability and polymorphism of the PCR amplification. The clarity and abundant polymorphism indicated that this system was stable and suitable for researching the genetic diversity ofP. infestans population.

Screening of Antagonistic Bacillus spp. Strains against Phytophthora infestans (Mont.) de Bary and Isolation of lipopeptide Substances

[ Objective] The paper was to screen the antagonistic Bacillus spp. strains against Phytophthora infestans （Mont.） de Bary and analyze its lipepeptide substances. [ Methods] Using flat isolation and culture method, the bacteria that could produce Bacillus spp. were isolated from rhizosphere soil, and carried out primary and secondary screening by confrontation culture. The lipopeptide substances of Y-3 strain with better antifungal effect were carried out I-IPLC analysis. [Result] Nine strains of bacteria had certain antagonistic effects against P. infestans in primary screening, Y-1 and Y-3 strains had significant inhibition effect against the growth of P. infestans in secondary screening, with inhibition rate higher than 80% ; the crude extract of lipepeptide produced by Y-3 strain had better antagonistic effect against P. infestans, with inhibition rate higher than 90%. HPLC analysis showed that the crude extract of lipepeptide mainly contained 8 components. [ Conclusion] The lipopeptide substances produced by Y-3 strain had significant inhibition effect, which had potential application value for biological control of potato late blight.

Analysis on Genotypic Differentiation of Phytophthora infestans by Using Random Amplified Polymorphic DNA (RAPD)

A total of 104 isolates including two Korean isolates and three Japanese isolates of Phytophthora infestans collected from Heilongjiang and Jilin Provinces from 2006 to 2008 were used to determine their mating types,metalaxyl resistance,and RAPD genotypes.All the isolates of P.infestans collected from Heilongjiang and Jilin Provinces belonged to A 1 mating type,and no A 2 mating type was detected.Frequencies of metalaxyl resistant isolates were 94.4%,47.8% and 75.0% in 2006,2007 and 2008,respectively.Accord...

Identification and Cloning of Differentially Expressed Genes Involved in the Interaction Between Potato and Phytophthora infestans using a Subtractive Hybridization and cDNA-AFLP Combinational Approach

Using a subtractive hybridization （SH）/cDNA-AFLP combinational approach, differentially expressed genes involved in the potato-Phytophthora infestans interaction were identified. These included genes potentially controlling pathogenesis or avr genes in P. infestans as well as those potentially involved in potato resistance or susceptibility to this pathogen. Forty-one differentially expressed transcript, derived fragments （TDFs）, resulting from the interaction, were cloned and sequenced. Two TDFs, suggested as potential pathogenicity factors, have sequence similarity to N-succinyl diaminopimelate aminotransferase and a transcriptional regulator, TetR family gene, respectively. Two other TDFs, suggested as potential avr genes, have sequence similarity to an EST sequence from Avr41Cf.41Avr91Cf- 9 and a P. infestans avirulence-associated gene, respectively. Genes＇ expression and origin were confirmed using Southern blots, Northern blots and qRT-PCR, he., potential resistance gene DL81 was induced at 12 hpi in the moderately resistant cultivar, whereas it was down-regulated as early as 6 hpi in the susceptible cultivar. On the other hand, DL21 was induced at 6 hpi （3.38-fold） in response to the highly aggressive isolate （US8） and strongly up-regulated thereafter （25.13-fold at 120 hpi.）, whereas it was only slightly up-regulated in response to the weakly aggressive isolate US11 （3.82-fold at 96 hpi）, suggesting its potential involvement as a susceptibility gene.

The lncRNA39896–miR166b–HDZs module affects tomato resistance to Phytophthora infestans

The yield and quality of tomatoes(Solanum lycopersicum)is seriously affected by Phytophthora infestans.The long non-coding RNA(lnc RNA)Sl-lnc RNA39896 is induced after P.infestans infection and was previously predicted to act as an endogenous target mimic(eTM)for the micro RNA Sl-miR166b,which function in stress responses.Here,we further examined the role of Sl-lncRNA39896 and Sl-mi R166b in tomato resistance to P.infestans.Sl-miR166b levels were higher in Sl-lnc RNA39896-knockout mutants than in wild-type plants,and the mutants displayed enhanced resistance to P.infestans.A six-point mutation in the region of Sl-lncRNA39896 that binds to Sl-miR166b disabled the interaction,suggesting that Sl-lnc RNA39896 acts as an e TM for Sl-mi R166b.Overexpressing Sl-miR166b yielded a similar phenotype to that produced by Sl-lncRNA39896-knockout,whereas silencing of Sl-mi R166b impaired resistance.We verified that Sl-miR166b cleaved transcripts of its target classⅢhomeodomain-leucine zipper genes Sl HDZ34and Sl HDZ45.Silencing of Sl HDZ34/45 decreased pathogen accumulation in plants infected with P.infestans.Additionally,jasmonic acid and ethylene contents were elevated following infection in the plants with enhanced resistance.Sl-lnc RNA39896is the first known lnc RNA to negatively regulate resistance to P.infestans in tomato.We propose a novel mechanism in which the lnc RNA39896–mi R166b–HDZ module modulates resistance to P.infestans.

Age-related Resistance and the Defense Signaling Pathway of Ph-3 Gene Against Phytophthora infestans in Tomatoes

Resistance(R) genes against plant pathogens often have age-related resistance(ARR) effects. However, the mechanism involved in this phenomenon remains unknown. In this paper, Solanum lycopersicum ‘CLN2037B' and S. pimpinellifolium ‘L3708' harboring the Ph-3 gene, as well as S. habrochaites ‘LA2099', ‘LA1777' and ‘LA1033' harboring quantitative trait loci(QTLs), were tested to investigate age-related resistance against late blight(LB; caused by Phytophthora infestans) in the three-leaf stage of the plants. The results demonstrated that the QTL-related LB resistance showed the same age-related resistance as the Ph-3-mediated resistance at the six- and nine-leaf stages compared with the three-leaf stage. This indicated that there is a common defense mechanism in tomatoes against P. infestans via ARR. In addition, we combined ethylene(ET), salicylic acid(SA) and jasmonic acid(JA) mutants with virus-induced gene silencing(VIGS) to study the Ph-3-dependent resistance signaling pathway. The results showed that ethylene and salicylic acid, but not jasmonic acid, are involved in the LB resistance mediated by the Ph-3 gene.

The Role of Autophagy during Development of the Oomycete Pathogen Phytophthora infestans

Phytophthora infestans is a highly destructive plant path- ogen that causes late blight disease in plants such as potato and tomato （Fry and Goodwin, 1997）. As a typical oomycete, the primary infective propagule of P. infestans is the sporangium, which can either germinate directly or release biflagellate zoospores that encyst and germinate on the host subsequently （Grenville-Briggs et al., 2005）. Due to abundant formation of the sporangia and prolific sporulation, P. infestans can spread quickly in the host tissues and cause extensive necrosis of the infected plants （Kamoun and Smart, 2005）.

Improvement of Polyacrylamide Gel Electrophorus in Phytophthora infeatans SSR Marker

[Objective] The paper was to improve polyacrylamide gel electrophorus in Phytophthora infeatans SSR Marker.[Method] With the disease sample of P.infeatans collected from Guyuan in Ningxia Province in 2009 as test material,its DNA was extracted and amplified with PCR,and its products were carried out polyacrylamide gel electrophoresis.[Result] 12% polyacrylamide gel electrophoresis was used to detect primers D13,G11 and PI02,and 8% polyacrylamide gel electrophoresis was used to detect primers PI4B,PI63,SSR4,SSR8 and SSR11,then 0.1% silver nitrate was used to stain,and an ideal electrophoresis and staining effect was obtained.[Conclusion] The electrophoresis and staining method suitable for P.infeatans SSR Marker established in the study had the characteristics of high sensitivity,simple operation and clear bands,which was an effective,simple and quick detection method.

Genome-wide analyses of mi RNAs in mycorrhizal plants in response to late blight and elucidation of the role of miR319c in tomato resistance

Tomato(Solanum lycopersicum), an economically important vegetable crop cultivated worldwide, often suffers massive financial losses due to Phytophthora infestans(P. infestans) spread and breakouts. Arbuscular mycorrhiza(AM) fungi mediated biocontrol has demonstrated great potential in plant resistance. However, little information is available on the regulation of mycorrhizal tomato resistance against P. infestans.Therefore, microRNAs(miRNAs) sequencing technology was used to analyse miRNA and their targets in the mycorrhizal tomato after P.infestans infection. Our study showed a lower severity of necrotic lesions in mycorrhizal tomato than in nonmycorrhizal controls. We investigated 35 miRNAs that showed the opposite expression tendency in mycorrhizal and nonmycorrhizal tomato after P. infestans infection when compared with uninfected P. infestans. Among them, miR319c was upregulated in mycorrhizal tomato leaves after pathogen infection. Overexpression of miR319c or silencing of its target gene(TCP1) increased tomato resistance to P. infestans, implying that miR319c acts as a positive regulator in tomato after pathogen infection. Additionally, we examined the induced expression patterns of miR319c and TCP1 in tomato plants exposed to salicylic acid(SA) treatment, and SA content and the expression levels of SA-related genes were also measured in overexpressing transgenic plants. The result revealed that miR319c can not only participates in tomato resistance to P. infestans by regulating SA content, but also indirectly regulates the expression levels of key genes in the SA pathway by regulating TCP1. In this study, we propose a novel mechanism in which the miR319c in mycorrhizal tomato increases resistance to P. infestans.

Inheritance Analysis and Identification of SSR Markers Linked to Late Blight Resistant Gene in Tomato

Late blight caused by Phytophthora infestans is the most serious disease of tomato production in China. Studies on the genetics of resistance and identification of molecular markers are very useful for breeding late blight resistant varieties. The objective of this paper was to study the inheritance of late blight resistance and identify simple sequence repeat （SSR） markers associated with resistance allele in tomato （Lycopersicon esculentum Mill）. The results came from an F2 progeny of 241 plants derived from a cross between 5~ inbred line that is susceptible to late blight and a resistant accession CLN2037E. The late blight responses of F2 plants were tested by artificially inoculation of detached-leaflets in plate and natural infection assayed under greenhouse conditions. Both methods showed that the resistance is dominant and inherited as monogenic trait. Genetic mapping and linkage analysis showed that the late blight resistance gene Ph-ROL was located on chromosome 9 with a genetic distance of 5.7 cM to the SSR marker TOM236.

Mancozeb： Its Value to the EU Potato Industry

Mancozeb is a multi-site fungicide used to control late blight （Phytophthora infestans） and early blight （Alternaria solani） in potatoes, and is currently due for re-approval as part of the EU Sustainable Use Directive---Authorisation of Plant Protection Products Regulation （EC） No 1107/2009. In order to understand its value to the EU potato industry, a stakeholder survey was conducted to understand the implications for P. infestans control and the impact on EU potato production if mancozeb was not available. In total, 319 growers and advisers were surveyed in 8 countries： France, Germany, Ireland, Netherlands, Greece, Italy, Spain and the UK, to determine how they use mancozeb, potential alternative control strategies and the implications of withdrawal. This study demonstrates that mancozeb is a widely used and cost-effective multisite active ingredient, which is a highly valued means of control for P. infestans. Based on the survey results, a loss of mancozeb would lead to a reduction in gross margin for potato producers in these countries of 787 million to 507 million depending on the level of P. infestans pressure. The combined impact of high cost of production and increased risks is likely to lead to reductions in the availability of EU potatoes and increased costs to consumers. In addition, an assessment was completed to determine the impact of mancozeb withdrawal on fungicide resistance development to single-site acting fungicides, finding that the loss of a multi-site active ingredient like mancozeb would severely compromise fungicide resistance management.

The Phytophthora nucleolar effector Pi23226 targets host ribosome biogenesis to induce necrotrophic cell death

Pathogen effectors target diverse subcellular organelles to manipulate the plant immune system.Although the nucleolus has emerged as a stress marker and several effectors are localized in the nucleolus,the roles of nucleolar-targeted effectors remain elusive.In this study,we showed that Phytophthora infestans infection of Nicotiana benthamiana results in nucleolar inflation during the transition from the biotrophic to the necrotrophic phase.Multiple P.infestans effectors were localized in the nucleolus:Pi23226 induced cell death in N.benthamiana and nucleolar inflation similar to that observed in the necrotrophic stage of infection,whereas its homolog Pi23015 and a deletion mutant(Pi23226DC)did not induce cell death or affect nucleolar size.RNA immunoprecipitation and individual-nucleotide-resolution UV crosslinking and immunoprecipitation sequencing analysis indicated that Pi23226 bound to the 30 end of 25S rRNA precursors,resulting in accumulation of unprocessed 27S pre-rRNAs.The nucleolar stress marker NAC082 was strongly upregulated under Pi23226-expressing conditions.Pi23226 subsequently inhibited global protein translation in host cells by interacting with ribosomes.Pi23226 enhanced P.infestans pathogenicity,indicating that Pi23226-induced ribosome malfunction and cell death were beneficial for pathogenesis in the host.Our results provide evidence for the molecular mechanism underlying RNA-binding effector activity in host ribosome biogenesis and lead to new insights into the nucleolar action of effectors in pathogenesis.