Assessment of changes in physiological and biochemical traits in four pistachio rootstocks under drought,salinity and drought + salinity stresses

In this study, 7-month-old UCB-1, Badami, Ghazvini and Kale-Ghouchi pistachio rootstocks were exposed to control, drought, salinity and drought + salinity environments for 60 d. Total chlorophyll and total carotenoid contents decreased in all cultivars under drought, salinity and drought + salinity stresses. Under drought and salinity stresses, alone or in combination, Na⁺ and Cl⁻ ions increased in all four pistachio rootstocks, while K⁺ ion decreased only in Ghazvini and Kaleh-Ghouchi cultivars. The enzyme activities of ascorbate peroxidase, polyphenol oxidase, catalase and guaiacol peroxidase increased in all cultivars when subjected to all three stresses with the exception of the ascorbate peroxidase activity in Kale-Ghouchi cultivar during drought stress. Oxidative stress parameters including electrolyte leakage, malondialdehyde, other aldehydes and hydrogen peroxide increased under all three stress conditions in all genotypes. The content of proline, total free amino acids and total soluble carbohydrates were enhanced under drought, salinity and drought + salinity stresses, whereas the protein content decreased in all pistachio rootstocks. In all evaluated traits, except for the K⁺ ion content and APX activity, the highest impacts was seen for drought + salinity > salinity > drought stresses, respectively. For the first time, we have proven that K⁺ ion content has a positive correlation with the ascorbate peroxidase, polyphenol oxidase, catalase and guaiacol peroxidase enzymes activities under drought + salinity stress. Finally, based on the bi-plot and cluster analyses, we have selected the UCB-1 > Badami > Ghazvini > Kale-Ghouchi cultivars as the most tolerant pistachio rootstocks under drought + salinity stress, respectively.     

Soil inoculum density of Verticillium dahliae and Verticillium wilt of olive in Lebanon

In the Mediterranean basin, Verticillium Wilt of Olive (VWO) is diffused throughout its range of cultivation, causing severe yield losses and tree mortality. The disease was reported in almost all the Mediterranean and Middle East countries, and in Lebanon it is of increasing significance also on many valuable crops. The disease has already been reported on potato, peach and almond in the Bekaa valley; however, to date no information is available about the incidence of VWO and the inoculum density of Verticillium dahliae microsclerotia in soil of the main agricultural areas of Lebanon. Results from the present investigations demonstrate a high V. dahliae frequency in soils (75.3%), coupled with a mean soil inoculum density of 17.0 MS g^?1, clearly indicating a great impact on the production of susceptible hosts in Lebanon, mainly in Bekaa region. Molecular method to assess the microsclerotia inoculum density in soil allowed the detection of a higher frequency of infested soils, as compared with the traditional plating, thus confirming its higher sensitivity. The overall Verticillium wilt prevalence in the inspected olive orchards was 46.2%, and the frequency of V. dahliae‐infected trees was 25.7%. The widespread presence of V. dahliae in all olive growing areas of Lebanon enforces the adoption of measures aimed at reducing the soil inoculum density before any new olive plantation, and the use of strong phytosanitary regulations to improve the certification schemes of propagating material.     

Detection of the defoliating and nondefoliating pathotypes of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in artificial and natural soils by nested PCR

In Spain, Verticillium wilt, caused by Verticillium dahliae, is the most important disease of cotton and olive. Isolates of V. dahliae infecting these crops can be classified into highly virulent, defoliating (D), and mildly virulent, nondefoliating (ND), pathotypes. Infested soil is the primary source of inoculum for Verticillium wilt epidemics in cotton and olive, and severity of disease relates to the prevailing V.dahliae pathotype. In this work we have adapted the use of previously developed primer pairs specific for D and ND V. dahliae for the detection of these pathotypes by nested PCR in artificial and natural soils. Success in the detection procedure depends upon efficiency in extracting PCR-quality DNA from soil samples. We developed an efficient DNA extraction method from microsclerotia infesting the soil that includes the use of acid washed sand during the grinding process and skimmed milk to avoid co-purification of Taq-polymerase inhibitors with DNA. The specific nested-PCR procedure effectively detected 10 or more microsclerotia per gram of soil. The detection procedure has proven efficient when used with a naturally infested soil, thus demonstrating usefullness of the diagnostic method for rapid and accurate assessment of soil contamination by V. dahliae pathotypes.     

Effect of Inoculum Density on Verticillium Wilt Incidence in Commercial Olive Orchards

Verticillium wilt, caused by Verticillium dahliae Kleb., is presently the most destructive disease of olive, particularly in Andalucía (southern Spain). ‘Picual’ and ‘Arbequina’ are the dominant cultivars being planted in Spain. Both cultivars are highly susceptible to the defoliating pathotype of V. dahliae when artificially inoculated by root‐dipping or stem injection. Conversely, ‘Arbequina’ is considered more resistant than ‘Picual’ based on field observations and farmer's experience. In this study, the differential reaction between of cultivars was confirmed by surveys of naturally infested orchards with different inoculum densities of the pathogen. The average percentage of affected olive trees of ‘Picual’ was 60.2%, while only 13.1% of trees of ‘Arbequina’ showed disease symptoms. Overall, the pathogen caused extensive wilting of branches and defoliation on the trees of ‘Picual’, whereas ‘Arbequina’‐infected trees showed chlorotic symptoms and slight defoliation. The relationship between inoculum density and disease incidence fit a logarithmic function for both cultivars. The percentage of affected trees of ‘Arbequina’ per year increased linearly (y = 0.3559x, R² = 0.5652, and P = 0.0195) with the inoculum density in the soil, whereas this relationship was not observed for the ‘Picual’. Planting density had no effect on disease incidence for any of the two cultivars.     

Effects of Drip Irrigation on Population of Verticillium dahliae in Olive Orchards

Verticillium wilt of olive (Olea europaea L.), caused by Verticillium dahliae, is nowadays the most serious olive disease in Spain. The disease increments are being observed particularly in young olive plantations, favoured by several factors including inadequate cultural practices and crop production intensification, such as irrigation. Thus, three olive orchards affected by Verticillium wilt, with disease incidence ranging 30–50%, were selected to determine if the drip irrigation could favour the increase of pathogen in soil. Pathogen in soil was quantified in wet zones around the drippers and in dry zones out of them. Inoculum density in all experiments was higher in wet than in dry areas. After 4 months of watering, soil pathogen population increased considerably in wet and dry areas but inoculum density remained higher in the wet soil.     

Salicylic Acid,as a Positive Regulator of Isochorismate Synthase,Reduces the Negative Effect of Salt Stress on Pistacia vera L. by Increasing Photosynthetic Pigments and Inducing Antioxidant Activity

Salinity, as a serious and prevalent abiotic stress, causes widespread crop losses by restricting plant growth and production throughout the world. In this study, the biochemical and molecular responses of the pistachio (Pistacia vera L.) plant were studied under NaCl and salicylic acid (SA) treatments using hydroponically grown salt tolerant (Ghazvini) and salt sensitive (Sarakhs) pistachio cultivars. NaCl treatment (250 mM) increased the production of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) and the activity of antioxidant enzymes in both cultivars. In the sensitive cultivar, the H₂O₂ content was higher than the tolerant cultivar, especially in the roots. SA application to both salt-stress-treated cultivars resulted in an increase in photosynthetic pigment contents and antioxidant enzyme activity and a decrease in the H₂O₂ and MDA contents. After NaCl treatment, the isochorismate synthase (ICS) gene was upregulated in Ghazvini which leads to an increase in the SA content of the salt tolerant pistachio cultivar. In contrast, the salt treatment downregulated the expression of the ICS gene in Sarakhs. The ICS gene expression was positively regulated by SA treatment under the salt stress condition. Our results suggest that Ghazvini has higher salinity tolerance than Sarakhs due to its higher antioxidant capacity, photosynthetic pigment content, and the cultivar-specific expression pattern of the ICS gene. In this study, the potential alleviative effects of SA on the adverse effect of salt stress in P. vera (Pistacia vera) were also identified and highlighted.

     

Relationships between rhizoplane and rhizosphere bacteria and verticillium wilt resistance in potato

Six cultivars and breeding lines of potato (Solanum tuberosum) differing in susceptibility to verticillium wilt caused by Verticillium dahliae were studied with respect to quantitative and qualitative differences in the bacterial flora of their soil and rhizosphere-rhizoplane. Although, no association was observed between the types of bacteria that inhabited the soil or roots of wilt resistant and susceptible cultivars, quantitative differences were evident. These differences provide the first direct evidence that potato genotypes can influence bacterial populations. Bacterial populations were 9–25-fold higher on roots than in the adjacent soil. As the plants aged, the total number of rootcolonizing bacteria increased between 15 and 245%. Pseudomonas spp. were the most abundant microbes in the soil and rhizosphere-rhizoplane. The bacteria antagonistic to V. dahliae in vitro were identified as members of the genera Bacillus, Pseudomonas, Flavobacterium, and Gluconobacter. A statistically significant trend was evident toward the association of antagonistic bacteria with the more resistant potato cultivars.     

Effects of arbuscular mycorrhizal fungi on photosystem II activity of three pistachio rootstocks under salt stress as probed by the OJIP-test

We applied chlorophyll a fluorescence as a biomarker to assess the growth response and PSII behavior and performance of three pistachio (Pistacia vera) rootstocks to different salt levels after inoculation with arbuscular mycorrhizal fungi Glomus mosseae and compared it with non-mycorrhizal plants (control). Our results confirmed the depressing effect of salt stress on mycorrhization extent and showed that the effect of salinity on colonization rate is completely under the influence of host plant. In this experiment, mycorrhizal symbiosis could enhance plants total dry mass (TDM), electron transfer on the donor and the acceptor side of PSII, decrease the energy dissipation and increase the comprehensive photosynthesis performance under salt stress as well as under normal conditions. We found that both donor and acceptor sides of PSII are the target sides under high salinity in pistachio rootstocks. We also found that performance index is the parameter that better reflects the responses of the studied rootstocks to progressive salt stress. Bane-baqi was less affected by salinity in terms of TDM followed by Sarakhs and Abareqi.     

The Influence Of Salinity On Verticillium dahliae In Stem Cuttings Of Five Olive Cultivars

Verticillium dahliae represents one of the main limiting factors in olive production in the Mediterranean countries. Increasing shortage of fresh water and land, increase the pressure on using alternative sources of marginal or saline water, and land previously cropped with V. dahliae host plants. The objective of the present study was to evaluate the influence of salinity on V. dahliae expression in olive stem cuttings. V. dahliae‐inoculated cuttings of cvs. Picual, Frantoio, Mansanillo and Barnea, showed higher senescence symptoms than their non‐inoculated controls. Colonization levels obtained in cv. Picual were significantly higher than in cv. Frantoio. Manzanillo was the most sensitive cultivar to salinity alone, with significant senescence symptoms in 4 and 6 dS/m NaCl treatments. When cv. Manzanillo was exposed to both salinity and V. dahliae, significantly higher senescence symptoms were obtained as compared with each of them separately. Senescence symptoms of cv. Picual exposed to V. dahliae, whether or not in combination with saline solutions, were significantly higher than those when cuttings were exposed to a saline solution alone. In cv. Frantoio, which is more resistant to salinity than the other cultivars, significantly high senescence symptoms were observed only in combination of V. dahliae and high saline concentration (8 dS/m). The fungal colonization index in cv. Manzanillo in high salinity (8 dS/m) was significantly higher than in the treatment without salt. In cv. Barnea, colonization index in 8 dS/m salinity was significantly higher than in the 4 dS/m concentration or control (fresh water). In conclusion, our findings demonstrate the interaction between V. dahliae and saline irrigation in various cultivars. Thus, stem cuttings could serve as an effective screening method in breeding olive clones for V. dahliae resistance, salt tolerance and their interaction.     

Characterization of Two Fungal Isolates from Cotton and Evaluation of their Potential for Biocontrol of Verticillium Wilt of Cotton

Two isolates (CVd‐WHw and CVn‐WHg) recovered from Verticillium‐wilt‐symptomatic cotton grown in Hubei Province of China were identified based on their morphology, growth characteristics in culture, specific amplification and identification of internal transcribed spacer (ITS) rDNA sequence. According to the morphological characteristics, specific PCR amplification and ITS sequences, CVd‐WHw was identified as V. dahliae and CVn‐WHg as Gibellulopsis nigrescens. In bioassays, the two isolates had significantly lower pathogenicity to cotton plant than V. dahliae isolate CVd‐AYb. Cotton pre‐inoculated with isolate CVn‐WHg or CVd‐WHw exhibited reduced disease indices of Verticillium wilt compared with those inoculated with CVd‐AYb alone. Cotton co‐inoculated with CVn‐WHg or CVd‐WHw and CVd‐AYb provided increased protection from subsequent CVd‐AYb inoculation. These results suggest that the two isolates have the potential to be developed as biocontrol agents for the control of Verticillium wilt in cotton. To our knowledge, this is the first report of a cross‐protection phenomenon using Gibellulopsis nigrescens against Verticillium wilt caused by V. dahliae on cotton.     

Colonization of Olive Inflorescences by Verticillium dahliae and its Significance for Pathogen Spread

Verticillium wilt of olive, caused by Verticillium dahliae Kleb., is the most severe disease affecting this crop in most olive growing countries. In this study, the presence of viable structures of V. dahliae in dried inflorescences from wilted olive shoots was investigated. The pathogen was found inside peduncles and flowers, by assessing the number of typical star‐shaped microsclerotial colonies formed onto the modified sodium polypectate agar medium. Microsclerotia of V. dahliae were observed inside the peduncles under the stereoscopic microscope. The presence of microsclerotia in these easily decomposable olive tissues shows that infected inflorescences can act as a source of inoculum for Verticillium wilt epidemics.     

Dynamic infection of Verticillium dahliae in upland cotton

• Verticillium wilt, an infection caused by the soilborne fungus Verticillium dahliae, is one of the most serious diseases in cotton. No effective control method against V. dahliae has been established, and the infection mechanism of V. dahliae in upland cotton remains unknown.
• GFP‐tagged V. dahliae isolates with different pathogenic abilities were used to analyse the colonisation and infection of V. dahliae in the roots and leaves of different upland cotton cultivars, the relationships among infection processes, the immune responses and the resistance ability of different cultivars against V. dahliae.
• Here, we report a new infection model for V. dahliae in upland cotton plants. V. dahliae can colonise and infect any organ of upland cotton plants and then spread to the entire plant from the infected organ through the surface and interior of the organ.
• Vascular tissue was found to not be the sole transmission route of V. dahliae in cotton plants. In addition, the rate of infection of a V. dahliae isolate with strong pathogenicity was notably faster than that of an isolate with weak pathogenicity. The resistance of upland cotton to Verticillium wilt was related to the degree of the immune response induced in plants infected with V. dahliae. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton.

     

Effects of cotton–maize rotation on soil microbiome structure

Verticillium wilt is a disastrous disease in cotton-growing regions in China. As a common management method, cotton rotation with cereal crops is used to minimize the loss caused by Verticillium dahliae. However, the correlation between soil microbiome and the control of Verticillium wilt under a crop rotation system is unclear. Therefore, three cropping systems (fallow, cotton continuous cropping, and cotton–maize rotation) were designed and applied for three generations under greenhouse conditions to investigate the different responses of the soil microbial community. The soil used in this study was taken from a long-term cotton continuous cropping field and inoculated with V. dahliae before use. Our results showed that the diversity of the soil bacterial community was increased under cotton–maize rotation, while the diversity of the fungal community was obviously decreased. Meanwhile, the structure and composition of the bacterial communities were similar even under the different cropping systems, but they differed in the soil fungal communities. Through microbial network interaction analysis, we found that Verticillium interacted with 17 bacterial genera, among which Terrabacter had the highest correlation with Verticillium. Furthermore, eight fungal and eight bacterial species were significantly correlated with V. dahliae. Collectively, this work aimed to study the interactions among V. dahliae, the soil microbiome, and plant hosts, and elucidate the relationship between crop rotation and soil microbiome, providing a new theoretical basis to screen the biological agents that may contribute to Verticillium wilt control.     

Water Stress and Verticillium Wilt Severity on Eggplant (Solanummelongena L.)

Verticillium wilt is one of the most destructive diseases of eggplant (Solanum melongena L.). Some researchers have reported that wilt was encouraged by sufficient soil humidity, while others stated that it was encouraged by drought. This study investigated the water stress effect on the severity of Verticillium wilt on eggplant, as it is reflected on yield, agronomic traits and fruit quality. Thus, eggplant seedlings cv. ‘Tsakoniki’ were transplanted in three rows, each with 20 plants, during the summer of 1995 and 1996 in a plastic greenhouse, at the Agricultural Research Center of Macedonia and Thrace. Ten of the plants in each row were inoculated with the fungus Verticillium dahliae, while the other 10 were used as controls. Rows were irrigated every 2, 4 or 6 days. Soil humidity was calculated before every irrigation in each row. The disease severity was estimated by the disease index (DI) as the combination product of leaf symptom index (LSI) and vascular discoloration index (VDI). In addition, the plant height, early and total commercial yields, fruit numbers of early and total commercial yields, plant weight, the above-ground plant weight, root weight, pH, total soluble solids and fruit brilliance plus colour intensity were measured. The effect of Verticillium wilt on plants irrigated every 2, 4 or 6 days was estimated by the correlation coefficient (r) between LSI and DI and the aforementioned characteristics. Verticillium wilt had a significant but negative effect on all of the measured or calculated characteristics. This effect, however, was independent of the irrigation applied. On average, the early commercial yield was reduced by 40.8% and the final commercial yield by 39.4%. The only quality characteristic that was affected significantly by irrigation was the fruit brilliance and colour intensity (r =?0.640 to ? 0.727, P ≤ 0.01). Finally, the irrigation frequency (every 2, 4 or 6 days) had a significant but negative effect on all of the characteristics measured on the control plants. The only exception was fruit quality. In conclusion, the combined effect of irrigation and Verticillium wilt infection significantly reduced the early and total production of eggplant and spoiled the fruit quality.     

Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection

Verticillium wilt of potato is caused by the fungus pathogen Verticillium dahliae. Present sRNA sequencing data revealed that miR482 was in response to V. dahliae infection, but the function in potato is elusive. Here, we characterized potato miR482 family and its putative role resistance to Verticillium wilt. Members of the potato miR482 superfamily are variable in sequence, but all variants target a class of disease‐resistance proteins with nucleotide binding site (NBS) and leucine‐rich repeat (LRR) motifs. When potato plantlets were infected with V. dahliae, the expression level of miR482e was downregulated, and that of several NBS‐LRR targets of miR482e were upregulated. Transgenic potato plantlets overexpressing miR482e showed hypersensitivity to V. dahliae infection. Using sRNA and degradome datasets, we validated that miR482e targets mRNAs of NBS‐LRR disease‐resistance proteins and triggers the production of trans‐acting (ta)‐siRNAs, most of which target mRNAs of defense‐related proteins. Thus, the hypersensitivity of transgenic potato could be explained by enhanced miR482e and miR482e‐derived ta‐siRNA‐mediated silencing on NBS‐LRR‐disease‐resistance proteins. It is speculated that a miR482‐mediated silencing cascade mechanism is involved in regulating potato resistance against V. dahliae infection and could be a counter defense action of plant in response to pathogen infection.     

Reaction of Cotton Cultivars and an F2 Population to Stem Inoculation with Isolates Verticillium dahliae

Four Verticillium dahliae isolates (V76, TS‐2, PH, and V44) were used in screening four cotton cultivars (Pima S‐7, Acala Prema, M‐315 and Acala 44). Pima S‐7 and Acala Prema gave the highest resistance reactions and Acala 44 was the most susceptible. Isolate V76 of V. dahliae was the most virulent. An interspecific cross between the resistant cv Pima S‐7 (Gossypium barbadense) and the susceptible cv. Acala 44 (G. hirsutum) was made and the F₂ population phenotyped for Verticillium wilt effect. Phenotyping of plant reaction to the disease was quantified by using a set of six growth parameters (number of healthy leaves, number of nodes, leaf weight, stem weight, leaf to stem ratio, and total shoot weight) measured 3 weeks after inoculation. The F₂ phenotypic distribution of these parameters suggests that distribution is towards resistance and polygenic. Transgressive segregation also was observed. The number of healthy leaves and total shoot weight were found to be the best indicators of resistance. Results obtained in this study will be useful to quantify resistance to V. dahliae and identify the best parameters to phenotype in genetic studies.     

Seedling vaccination by stem injecting a conidial suspension of F2, a non-pathogenic Fusarium oxysporum strain, suppresses Verticillium wilt of eggplant

Several bacterial and fungal strains have been evaluated as biocontrol agents (BCAs) against Verticillium dahliae. In these studies, the BCAs were applied as a root drenching inoculum; however, this application method may have an adverse effect on the native, beneficial for the plants, microbial community. In the present study, it was evaluated whether endophytic application by stem injecting a conidial suspension of the non pathogenic Fusarium oxysporum F2 strain, isolated from a V. dahliae suppressive compost amendment, could reduce significantly Verticillium wilt symptom development in eggplants. It was revealed that stem injection of F2 seven days before transplanting the seedlings to soil infested by V. dahliae microsclerotia resulted in reduced disease severity compared to the control treatment. To visualise F2 ramification into the plant vascular system eggplant stems were injected with an EGFP transformed F2 strain. It was shown that F2 colonises effectively the plant vascular tissues over a long period of time as it was assessed by F2 DNA levels. In parallel, qPCR analysis showed that the application of F2 reduced significantly the amount of V. dahliae DNA in the stem tissues compared to the control treatment.     

The interactions of potato cyst nematodes and Verticillium dahliae on early and maincrop potato cultivars

The effects of potato cyst nematodes (Globodera rostochiensis and G. pallida) on the growth of early and later maturing potato cultivars, with or without the H, resistance gene, were compared in pot experiments and the effects of introducing Verticillium dahliae into the system were studied. The influence of the nematodes on root function and structural integrity was inferred from their effects on calcium and water uptake. Early maturing cultivars were less tolerant of nematode attack than later maturing ones, and those with the H₁ resistance gene were more tolerant of G. rostochiensis than G. pallida. When V. dahliae was introduced, Verticillium wilt symptoms appeared much earlier on the early than the maincrop cultivars. Also, Maris Anchor (first early) died before Maris Peer (second early) and death of both when infected by nematodes and fungus was much earlier than uninfected control plants or plants infected with G. rostochiensis only. Maris Peer infected by both organisms snowed symptoms much more severe than when infected by either organism alone, whereas Maris Anchor was almost as severely affected by V. dahliae alone as both organisms together. Pentland Javelin (first early) developed Verticillium wilt symptoms several weeks later than other cultivars and the addition of nematodes failed to increase the severity of the symptoms. These differences in cultivar susceptibility to the combination of nematodes and fungus may be due to the nematodes causing different degrees of trauma in the roots of the three cultivars: measuring calcium uptake per unit water uptake showed that nematodes dramatically increase calcium uptake but that the proportional change is least in Pentland Javelin. If this is a reflection of less damage by nematodes to the endodermis of this cultivar, it may explain the greater resistance of its roots to penetration by V. dahliae.