Effect of Acinetobacter sp on metalaxyl degradation and metabolite profile of potato seedlings (Solanum tuberosum L.) alpha variety

One of the most serious diseases in potato cultivars is caused by the pathogen Phytophthora infestans, which affects leaves, stems and tubers. Metalaxyl is a fungicide that protects potato plants from Phytophthora infestans. In Mexico, farmers apply metalaxyl 35 times during the cycle of potato production and the last application is typically 15 days before harvest. There are no records related to the presence of metalaxyl in potato tubers in Mexico. In the present study, we evaluated the effect of Acinetobacter sp on metalaxyl degradation in potato seedlings. The effect of bacteria and metalaxyl on the growth of potato seedlings was also evaluated. A metabolite profile analysis was conducted to determine potential molecular biomarkers produced by potato seedlings in the presence of Acinetobacter sp and metalaxyl. Metalaxyl did not affect the growth of potato seedlings. However, Acinetobacter sp strongly affected the growth of inoculated seedlings, as confirmed by plant length and plant fresh weights which were lower in inoculated potato seedlings (40% and 27%, respectively) compared to the controls. Acinetobacter sp also affected root formation. Inoculated potato seedlings showed a decrease in root formation compared to the controls. LC-MS/MS analysis of metalaxyl residues in potato seedlings suggests that Acinetobacter sp did not degrade metalaxyl. GC-TOF-MS platform was used in metabolic profiling studies. Statistical data analysis and metabolic pathway analysis allowed suggesting the alteration of metabolic pathways by both Acinetobacter sp infection and metalaxyl treatment. Several hundred metabolites were detected, 137 metabolites were identified and 15 metabolic markers were suggested based on statistical change significance found with PLS-DA analysis. These results are important for better understanding the interactions of putative endophytic bacteria and pesticides on plants and their possible effects on plant metabolism.     

Effect of proteinaceous proteinase inhibitors from potato tubers on the growth and development of phytopathogenic microorganisms

We studied the effect of two proteins, PSPI-21 and PKSI, on the growth and development of phytopathogenic microorganisms (Phytophthora infestans oomycete and Fusarium culmorum fungus). Both proteins were isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and served as inhibitors of serine proteinases. These proteins differed in the ability to inhibit growth of Phytophthora infestans oomycete and Fusarium culmorum fungus. PSPI-21 was the most potent in modulating the growth of oomycete mycelium. PKSI primarily affected the growth of the fungal mycelium. The proteins under study induced complete destruction of oomycete zoospores and partial destruction of fungal macroconidia. Our results suggest that these proteins are involved in the protection of potato plants from phytopathogenic microorganisms.     

Purification of an isoform of patatin with antimicrobial activity against Phytophthora infestans.

Phytophthora infestans (Mont.) de Bary is infamous as the causal agent of the late blight epidemic contributing to the Irish potato famine of the mid 19th century and remains agriculture's most destructive disease as new mutations and migrations confound control measures. In efforts to develop resistant varieties, a somatic hybrid (the Wisconsin J series) between potato (Solanum tuberosum) and a wild relative (Solanum bulbocastanum) has been found to convey durable resistance against the pathogen. We screened the total protein (100 microg ml(-1)) of somatic hybrid varieties J138, J138A12, J101K12, J103K12, and J101K9 for in vitro spore germination inhibition of P. infestans. Since J138 exhibited maximum inhibition at 150 microg ml(-1) in comparison to other varieties, we purified a 40 kD protein from J138 tubers by assaying its ability to inhibit spore germination in P. infestans spores. The highly purified protein was able to inhibit P. infestans spore germination by 70% at the 2.5 microg ml(-1) concentration. The N-terminal sequence of this protein was found to have exact amino acid homology to patatin, the major storage protein of potato tubers. The inhibitory protein has the same molecular weight as patatin and cross-reacts with patatin antibodies. The infection of J138 plants with spores of P. infestans under greenhouse conditions showed that patatin is expressed in stem tissue 72 h after the plant is inoculated with field isolates of P. infestans (US8). In this communication, we report the purification, characterization and antifungal activity against spores of P. infestans of patatin-J from potato tubers.     

Development of PCR primers from internal transcribed spacer region 2 for detection of Phytophthora species infecting potatoes.

We developed PCR primers and assay methods to detect and differentiate three Phytophthora species which infect potatoes and cause late blight (Phytophthora infestans) and pink rot (P. erythroseptica and P. nicotianae) diseases. Primers based on sequence analysis of internal transcribed spacer region 2 of ribosomal DNA produced PCR products of 456 bp (P. infestans), 136 bp (P. erythroseptica), and 455 bp (P. nicotianae) and were used to detect the pathogens in potato leaf (P. infestans) and tuber (P. infestans, P. erythroseptica, and P. nicotianae) tissue with a sensitivity of 1 to 10 pg of DNA. Leaf and tuber tissue were processed for PCR by a rapid NaOH method as well as a method based on the use of commercially available ion-exchange columns of P. infestans primers and the rapid NaOH extraction method were used to detect late blight in artificially and naturally infected tubers of potato cultivar Red LaSoda. In sampling studies, P. infestans was detected by PCR from artificially infected tubers at 4 days postinoculation, before any visible symptoms were present. The PCR assay and direct tissue extraction methods provide tools which may be used to detect Phytophthora pathogens in potato seedlots and storages and thus limit the transmission and spread of new, aggressive strains of P. infestans in U.S. potato-growing regions.     

Transgenic Potatoes Expressing a Novel Cationic Peptide are Resistant to Late Blight and Pink Rot

Potato is the world's largest non-cereal crop. Potato late blight is a pandemic, foliar wasting potato disease caused by Phytophthora infestans, which has become highly virulent, fungicide resistant, and widely disseminated. Similarly, fungicide resistant isolates of Phytophthora erythroseptica, which causes pink rot, have also become an economic scourge of potato tubers. Thus, an alternate, cost effective strategy for disease control has become an international imperative. Here we describe a strategy for engineering potato plants exhibiting strong protection against these exceptionally virulent pathogens without deleterious effects on plant yield or vigor. The small, naturally occurring antimicrobial cationic peptide, temporin A, was N-terminally modified (MsrA3) and expressed in potato plants. MsrA3 conveyed strong resistance to late blight and pink rot phytopathogens in addition to the bacterial pathogen Erwinia carotovora. Transgenic tubers remained disease-free during storage for more than 2 years. These results provide a timely, sustainable, effective, and environmentally friendly means of control of potato diseases while simultaneously preventing storage losses.     

马铃薯晚疫病生防木霉菌的筛选及鉴定

采用马铃薯活体筛选法从268株木霉菌中筛选获得两株对致病疫霉有较强抑菌活性的木霉菌株R-5和T-15。这两株木霉菌代谢液可抑制病原菌生长及孢子囊萌发。温室防病试验发现,接种两株木霉菌可以减轻晚疫病的发生。田间试验进一步证明,两株木霉菌对晚疫病具有良好的田间防治效果,防效分别达到了72.4%和70.0%。经分子生物学方法鉴定,两株木霉菌分别为拟康氏木霉和棘孢木霉。实验构建的以活体筛选为基础的生防木霉菌筛选方法是一种可行高效的生防木霉菌筛选方式。     

Inhibition of fungal and bacterial plant pathogens by synthetic peptides: in vitro growth inhibition, interaction between peptides and inhibition of disease progression

Four synthetic cationic peptides, pep6, pep7, pep11 and pep20, were tested alone and in combinations for their antimicrobial activities against economically important plant pathogenic fungi (Phytophthora infestans and Alternaria solani) and bacteria (Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica). In in vitro studies, P. infestans and A. solani were inhibited by all four peptides, while E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica were inhibited only by pep11 and pep20. All peptides completely inhibited P. infestans and A. solani on potato leaves and P. infestans on tubers at concentrations comparable to the in vitro IC50 (effective concentration for 50% growth inhibition) values, suggesting that these peptides are more potent in preventing infection than in inhibiting hyphal growth in vitro. Microscopic observations of P. infestans and A. solani when treated with these peptides revealed hyphal anomalies. In tuber-infectivity assays, pep11 and pep20 reduced bacterial softrot symptoms by 50% at 2.0 to 2.30 microM and by 100% at 20 microM. In assays involving two-way combinations of these peptides, growth inhibitions of fungi and bacteria by the combinations were no more than the sum of growth inhibitions by each peptide when used alone, indicating that they act additively. pep11 and pep20 are not phytotoxic to potato plants at 200 microM. With strong and broad-spectrum antimicrobial activities of pep11 and pep20 against fungi and bacteria, and with no antagonistic activities, the expression of these peptides in transgenic potato plants could lead to enhanced disease resistance against these pathogens.     

Cloning, expression, and sequence conservation of pathogenesis-related gene transcripts of potato

Treatment of potato tuber disks with arachidonic acid elicits the accumulation of several mRNAs. cDNA clones corresponding to two of these mRNAs were isolated and characterized. Nucleotide sequence analysis reveals that both clones (pSTH-2 and pSTH-21) contain an open-reading frame coding for a 155-amino acid polypeptide. The polypeptides encoded by the two clones differ by only six amino acids and show a high degree of similarity with PR protein sequences from pea (approximately 42%) and parsley (approximately 37%). mRNAs corresponding to the two potato cDNA clones also accumulate in Solanum chacoense and in tomato following elicitor treatment. Maximum accumulation of the mRNAs corresponding to the two cDNA clones is reached 24 hr after elicitor treatment of the tuber disks. pSTH-2-related mRNAs also accumulate in tubers after wounding or treatment with eicosapentaenoic acid and are detected in potato and tomato leaves treated with a Phytophthora infestans mycelium homogenate. The presence of these conserved genes in species from three plant families and the similarity of their induction pattern suggest an important function during the plant defense response.     

Salicylic acid is important for basal defense of Solanum tuberosum against Phytophthora infestans

The importance of the signaling compound salicylic acid for basal defense of potato (Solanum tuberosum L. cv. Désirée) against Phytophthora infestans, the causal agent of late blight disease, was assessed using transgenic NahG potato plants which are unable to accumulate salicylic acid. Although the size of lesions caused by P. infestans was not significantly different in wild-type and transgenic NahG plants, real-time polymerase chain reaction analyses revealed a drastic enhancement of pathogen growth in potato plants depleted of salicylic acid. Increased susceptibility of NahG plants correlated with compromised callose formation and reduced early defense gene expression. NahG plants pretreated with the salicylic acid analog 2,6-dichloro-isonicotinic acid allowed pathogen growth to a similar extent as did wild-type plants, indicating that salicylic acid is an important compound required for basal defense of potato against P. infestans.     

Exoproteinases of the oomycete Phytophthora infestans

When grown in a medium containing heat-stable potato tuber proteins, the oomycete Phytophthora infestans (Mont.) de Bary produces a set of exoproteinases active at neutral and mildly basic pH values. These extracellular proteinases have been shown by SDS-PAGE with the presence of gelatin to include at least six components differing in molecular weight. Inhibitory analysis and study of the effects of the enzymes on various synthetic substrates show that the culture liquid of P. infestans contains mainly serine proteinases specific to trypsin and subtilisin and metalloproteinases. Their activity is suppressed by proteinase-inhibitor proteins from potato tubers. It is suggested that P. infestans exoproteinases may be the metabolic target for natural proteinase inhibitors from potato.     

Rewiring mitogen-activated protein kinase cascade by positive feedback confers potato blight resistance

Late blight, caused by the notorious pathogen Phytophthora infestans, is a devastating disease of potato (Solanum tuberosum) and tomato (Solanum lycopersicum), and during the 1840s caused the Irish potato famine and over one million fatalities. Currently, grown potato cultivars lack adequate blight tolerance. Earlier cultivars bred for resistance used disease resistance genes that confer immunity only to some strains of the pathogen harboring corresponding avirulence gene. Specific resistance gene-mediated immunity and chemical controls are rapidly overcome in the field when new pathogen races arise through mutation, recombination, or migration from elsewhere. A mitogen-activated protein kinase (MAPK) cascade plays a pivotal role in plant innate immunity. Here we show that the transgenic potato plants that carry a constitutively active form of MAPK kinase driven by a pathogen-inducible promoter of potato showed high resistance to early blight pathogen Alternaria solani as well as P. infestans. The pathogen attack provoked defense-related MAPK activation followed by induction of NADPH oxidase gene expression, which is implicated in reactive oxygen species production, and resulted in hypersensitive response-like phenotype. We propose that enhancing disease resistance through altered regulation of plant defense mechanisms should be more durable and publicly acceptable than engineering overexpression of antimicrobial proteins.     

Tracking historic migrations of the Irish potato famine pathogen,Phytophthora infestans

The plant pathogen Phytophthora infestans causes late blight, a devastating disease on potato that led to the Irish potato famine during 1845-1847. The disease is considered a reemerging problem and still causes major epidemics on both potato and tomato crops worldwide. Theories on the origin of the disease based on an examination of the genetic diversity and structure of P. infestans populations and use of historic specimens to understand modern day epidemics are discussed.     

Activity of a proteinaceous polygalacturonase inhibitor in potato plants

The activity of a protein inhibitor of polygalacturonase (PIPG) was studied in potato tubers during storage and in potato leaves and stems during vegetation. The activity of PIPG in tubers varied from between seasons. The activity of PIPG during dormancy changed depending on the storage stage and temperature. As a rule, it was higher in etiolated sprouts than in the tubers. The activity of PIPG was slightly higher in leaves of adult vegetating plants than in stems and decreased by the end of vegetation. These changes in the activity of PIPG are suggested to be associated with changes in the growth rate.     

Inhibitory effect of a defensin gene from the Andean crop maca (Lepidium meyenii) against Phytophthora infestans

In this study, we report the isolation of a defensin gene, lm-def, isolated from the Andean crop 'maca' (Lepidium meyenii) with activity against the pathogen Phytophthora infestans responsible of late blight disease of the potato and tomato crops. The lm-def gene has been isolated by polymerase chain reaction (PCR) using degenerate primers corresponding to conserved regions of 13 plant defensin genes of the Brassicaceae family assuming that defensin genes are highly conserved among cruciferous species. The lm-def gene belongs to a small multigene family of at least 10 members possibly including pseudogenes as assessed by genomic hybridization and nucleotide sequence analyses. The deduced mature Lm-Def peptide is 51 amino acids in length and has 74-94% sequence identity with other plant defensins of the Brassicaceae family. The Lm-Def peptide was produced as a fusion protein using the pET-44a expression vector and purified using an immobilized metal ion affinity chromatography. The recombinant protein (NusA:Lm-Def) exhibited in vitro activity against P. infestans. The NusA:Lm-Def protein caused growth inhibition and hyphal damage at concentration not greater than 0.4 microM. In contrast, the NusA protein alone expressed and purified similarly did not show any activity against P. infestans. Therefore, these results indicate that the lm-def gene isolated from maca belong to the plant defensin family with activity against P. infestans. Its expression in potato, as a transgene, might help to control the late blight disease caused by P. infestans with the advantage of being of plant origin.     

Activity of a protein inhibitor of polygalacturonase in potato plants

The activity of a protein inhibitor of polygalacturonase (PIPG) was studied in potato tubers during storage and in potato leaves and stems during vegetation. The activity of PIPG in tubers varied between seasons. The activity of PIPG during dormancy changed depending on the storage stage and temperature. As a rule, it was higher in etiolated sprouts than in the tubers. The activity of PIPG was slightly higher in leaves of adult vegetating plants than in stems and decreased by the end of vegetation. These changes in the activity of PIPG are suggested to be associated with changes in the growth rate.     

Changes in the Activity of Polygalacturonase-Inhibiting Protein during Potato Development

The activity of a polygalacturonase-inhibiting protein was determined in growing potato plants and in stored potato tubers. The activity in leaves was higher than in stems, and it decreased by the end of the vegetative season. During the dormancy period, the inhibitory activity in tubers also changed. In the sprouting tubers, it was somewhat lower than in the nonsprouting ones, and, in sprouts, it was usually higher than in tubers. Both the plant polygalacturonase and the polygalacturonase secreted by phytopathogenic fungi after their penetration in plant tissues can serve as inhibitor's targets. Therefore, the inhibitor seems to control the resistance of plants to infection by particular pathogens, and this resistance is characteristic of definite developmental stages.