Yield and yield components of hybrid corn (Zea mays L.) as affected by mycorrhizal symbiosis and zinc sulfate under drought stress

With respect to the significance of improving hybrid corn performance under stress, this experiment was conducted at the Islamic Azad University, Arak Branch, Iran. A complete randomized block design with three levels of irrigations (at 100%, 75% and 50% crop water requirement), two levels of arbuscular mycorrhizal (AM) fungi (Glumus intraradisis) (including control), and three levels of zinc (Zn) sulfate (0, 25 and 45 kg ha⁻¹), was performed. Results of the 2-year experiments indicated that irrigation treatment significantly affected corn yield and its components at P = 1%. AM fungi and increasing Zn levels also resulted in similar effects on corn growth and production. Although AM fungi did not significantly affect corn growth at the non-stressed irrigation treatment, at moderate drought stress AM fungi significantly enhanced corn quality and yield relative to the control treatment. The combined effects of AM fungi and Zn sulfate at 45 kg ha⁻¹ application significantly affected corn growth and production. In addition, the tripartite treatments significantly enhanced corn yield at P = 1%. Effects of Zn and AM fungi on plant growth under drought stress is affected by the stress level.     

Plant growth stimulation of wheat (Triticum aestivum L.) by inoculation of salinity tolerant Azotobacter strains

Five salinity tolerant Azotobacter strains i.e., ST3, ST6, ST9, ST17 and ST24 were obtained from saline soils. These Azotobacter strains were used as inoculant for wheat variety WH157 in earthen pots containing saline soil under pot house conditions, using three fertilizer treatment doses i.e., control (no fertilizer, no inoculation), 90 Kg N ha⁻¹ and 120 Kg N ha⁻¹. Inoculation with salinity tolerant Azotobacter strains caused significant increase in total nitrogen, biomass and grain yield of wheat. Maximum increase in plant growth parameters were obtained after inoculation with Azotobacter strain ST24 at fertilization dose of 120 kg N ha⁻¹ and its inoculation resulted in attaining 89.9 cms plant height, 6.1 g seed yield, 12.0 g shoot dry weight and 0.7 % total nitrogen. The survival of Azotobacter strain ST24 in the soil was also highest in all the treatments at 30, 60 and 90 days after sowing (DAS). However, the population of Azotobacter decreased on 90 DAS as compared to counts observed at 60 DAS at all the fertilization treatments.     

Direct and indirect influences of arbuscular mycorrhizal fungi on phosphorus uptake by two root hemiparasitic Pedicularis species: do the fungal partners matter at low colonization levels?

Background and Aims

Because most parasitic plants do not form mycorrhizal associations, the nutritional roles of arbuscular mycorrhizal (AM) fungi in them have hardly been tested. Some facultative root hemiparasitic Pedicularis species form AM associations and hence are ideal for testing both direct and indirect effects of AM fungi on their nutrient acquisition. The aim of this study was to test the influence of AM inoculation on phosphorus (P) uptake by Pedicularis rex and P. tricolor.

Methods

³²P labelling was used in compartmented pots to assess the contribution of the AM pathway and the influence of AM inoculation on P uptake from a host plant into the root hemiparasites. Laboratory isolates of fungal species (Glomus mosseae and G. intraradices) and the host species (Hordeum vulgare ‘Fleet’) to which the two Pedicularis species showed obvious responses in haustorium formation and growth in previous studies were used.

Key Results

The AM colonization of both Pedicularis spp. was low (<15 % root length) and only a very small proportion of total plant P (<1 %) was delivered from the soil via the AM fungus. In a separate experiment, inoculation with AM fungi strongly interfered with P acquisition by both Pedicularis species from their host barley, almost certainly because the numbers of haustoria formed by the parasite were significantly reduced in AM plants.

Conclusions

Roles of AM fungi in nutrient acquisition by root parasitic plants were quantitatively demonstrated for the first time. Evidence was obtained for a novel mechanism of preventing root parasitic plants from overexploiting host resources through AM fungal-induced suppression of the absorptive structures in the parasites.     

Influence of exogenous application of glycinebetaine on antioxidative system and growth of salt-stressed soybean seedlings (Glycine max L.)

Glycinebetaine is one of the most competitive compounds which play an important role in salt stress in plants. In this study, the enhanced salt tolerance in soybean (Glycine max L.) by exogenous application of glycinebetaine was evaluated. To improve salt tolerance at the seedling stage, GB was applied in four different concentrations (0, 5, 25 and 50 mM) as a pre-sowing seed treatment. Salinity stress in the form of a final concentration of 150 mM sodium chloride (NaCl) over a 15 day period drastically affected the plants as indicated by increased proline, MDA and Na⁺ content of soybean plants. In contrast, supplementation with 50 mM GB improved growth of soybean plants under NaCl as evidenced by a decrease in proline, MDA and Na⁺ content of soybean plants. Further analysis showed that treatments with GB, resulted in increasing of CAT and SOD activity of soybean seedlings in salt stress. We propose that the role of GB in increasing tolerance to salinity stress in soybean may result from either its antioxidant capacity by direct scavenging of H₂O₂ or its role in activating CAT activity which is mandatory in scavenging H₂O₂.     

Shoot culture of Bacopa monnieri: standardization of explant,vessels and bioreactor for growth and antioxidant capacity

Standardization of biomass production in different vessels and bioreactor using explants and media for growth, total phenolic content and antioxidant capacity of shoot culture of Bacopa monnieri is described. Maximum number of shoots per explant, higher explants response irrespective of the type of explants, and higher shoot length was obtained on MS medium containing BAP (2.5 mg l⁻¹) and IAA (0.01 mg l⁻¹) with 3 % sucrose. This medium was selected by varying BAP concentration and recorded optimal for shoot culture on gelled medium. The condition of 0.5 cm explant size and 20 explant/40 ml (1 explant/2 ml) was optimal for high explant response, number of shoots per explant regenerated and shoots length. Among the different vessels used, maximum growth index was achieved in Growtek bioreactor (10.0) followed by magenta box (9.16), industrial glass jar (7.7) and conical flask (7.2). The cultures grown in conical flask (100 ml) were used as control. The total phenolic content and antioxidant capacity of in vitro grown plants was higher to that recorded for in vivo material. Among in vitro regenerated plants, the activity was maximal in the tissues grown in 250 ml conical flask. The most critical function for vessels is to support the optimum profusion (growing area for maximum growth) of shoots and for B. monnieri, Growtek bioreactor supported 1980 shoots l⁻¹ medium as compared to control (938 shoots l⁻¹). Growtek bioreactor was considered effective system to produce B. monnieri biomass in culture without loss of antioxidant properties.     

Effect of Sclerotinia sclerotiorum on the disease development,growth, oil yield and biochemical changes in plants of Mentha arvensis

Experiment was carried out to determine the effect of Sclerotinia sclerotiorum on the disease development, growth, oil yield and biochemical changes in the plants of Mentha arvensis. With the increase in initial inoculum levels of S. sclerotiorum a corresponding decrease in plant fresh and dry weights were recorded. The maximum reduction in the shoot-roots/suckers fresh weight and shoot-roots/suckers dry weights (39.8%, 43.6%, 40.3% and 42.9%), respectively, was observed at the highest initial inoculum level of 12 g fungal mycelium/5 kg soil as compared to uninoculated control. The infection of roots and suckers due to S. sclerotiorum increased with increasing initial inoculum levels. At the lowest initial inoculum (1.0 g mycelium/5 kg soil), infection was observed 18.0% and at the highest (12 g mycelium/5 kg soil), it was 80.2%. Significant (P ⩽ 0.01) reduction in oil yield, total chlorophyll, total phenol and total sugar content of M. arvensis plants was observed at the lowest inoculum level as compared to uninoculated control.     

Combined inoculation with Glomus intraradices and Rhizobium tropici CIAT899 increases phosphorus use efficiency for symbiotic nitrogen fixation in common bean (Phaseolus vulgaris L.)

This study compared the response of common bean (Phaseolus vulgaris L.) to arbuscular mycorrhizal fungi (AMF) and rhizobia strain inoculation. Two common bean genotypes i.e. CocoT and Flamingo varying in their effectiveness for nitrogen fixation were inoculated with Glomus intraradices and Rhizobium tropici CIAT899, and grown for 50 days in soil–sand substrate in glasshouse conditions. Inoculation of common bean plants with the AM fungi resulted in a significant increase in nodulation compared to plants without inoculation. The combined inoculation of AM fungi and rhizobia significantly increased various plant growth parameters compared to simple inoculated plants. In addition, the combined inoculation of AM fungi and rhizobia resulted in significantly higher nitrogen and phosphorus accumulation in the shoots of common bean plants and improved phosphorus use efficiency compared with their controls, which were not dually inoculated. It is concluded that inoculation with rhizobia and arbuscular mycorrhizal fungi could improve the efficiency in phosphorus use for symbiotic nitrogen fixation especially under phosphorus deficiency.     

Penicillium ochrochloron MTCC 517 chitinase: An effective tool in commercial enzyme cocktail for production and regeneration of protoplasts from various fungi

Penicillium ochrochloron MTCC 517 is a potent producer of chitinolytic enzymes. Novozyme 234, traditional enzyme cocktail for protoplast generation is not available in the market. So, new enzyme cocktail is prepared for protoplast formation from various filamentous fungi which consists of 5 mg ml⁻¹ lysing enzymes from Trichoderma harzianum, 0.06 mg ml⁻¹ β-glucuronidase from Helix pomatia and 1 mg ml⁻¹P. ochrochloron chitinase. The greatest number of protoplasts could be produced from most of the fungi in 0.8 M sorbitol and by incubation for about 2 h at 37 °C, but the number was decreased by incubation for more than 3 h. About twice as many protoplasts were produced from different species of fungi by involvement of P. ochrochloron chitinase than with combined commercial enzymes.     

Mild ammonium stress increases chlorophyll content in Arabidopsis thaliana

Nitrate (NO₃⁻) and ammonium (NH₄⁺) are the main forms of nitrogen available in the soil for plants. Excessive NH₄⁺ accumulation in tissues is toxic for plants and exclusive NH₄⁺-based nutrition enhances this effect. Ammonium toxicity syndrome commonly includes growth impairment, ion imbalance and chlorosis among others. In this work, we observed high intraspecific variability in chlorophyll content in 47 Arabidopsis thaliana natural accessions grown under 1 mM NH₄⁺ or 1 mM NO₃⁻ as N-source. Interestingly, chlorophyll content increased in every accession upon ammonium nutrition. Moreover, this increase was independent of ammonium tolerance capacity. Thus, chlorosis seems to be an exclusive effect of severe ammonium toxicity while mild ammonium stress induces chlorophyll accumulation.     

Relationship between Meloidogyne arenaria and Aflatoxin Contamination in Peanut

Damaged and developing kernels of peanut (Arachis hypogaea) are susceptible to colonization by fungi in the Aspergillus flavus group which, under certain conditions, produces aflatoxins prior to harvest. Our objective was to determine whether infection of peanut roots and pods by Meloidogyne arenaria increases aflatoxin contamination of the kernels when peanut is subjected to drought stress. The experiment was a completely randomized 2-x-2 factorial with 6 replicates/treatment. The treatment factors were nematodes (plus and minus M. arenaria) and fungus (plus and minus A. flavus inoculum). The experiment was conducted in 2001 and 2002 in microplots under an automatic rain-out shelter. In treatments where A. flavus inoculum was added, aflatoxin concentrations were high (> 1,000 ppb) and not affected by nematode infection; in treatments without added fungal inoculum, aflatoxin concentrations were greater (P ≤ 0.05) in kernels from nematode-infected plants (1,190 ppb) than in kernels from uninfected plants (79 ppb). There was also an increase in aflatoxin contamination of kernels with increasing pod galling (r² = 0.83 in 2001, r² = 0.43 in 2002; P ≤ 0.04). Colonization of kernels by A. flavus increased with increasing pod galling (r² = 0.18; P = 0.04) in 2001 but not in 2002. Root-knot nematodes may have a greater role in enhancing aflatoxin contamination of peanut when conditions are not optimal for growth and aflatoxin production by fungi in the A. flavus group.     

Penconazole induced changes in photosynthesis, ion acquisition and protein profile of Mentha pulegium L. under drought stress

Effect of penconazole (PEN) treatment on drought-stressed Mentha pulegium L. plants was investigated. Six weeks after sowing, seedlings were grown under soil moisture corresponding to 100, 75, 50 and 25 % field capacity (FC) with or without PEN (15 mg l⁻¹) for 4 weeks. Results showed that the seedlings at 75 % FC showed maximum growth and water supply lower than 75 % FC was the threshold of drought-initiated negative effects on seedling growth. Drought stress significantly induced proline and carbohydrate contents and the decreased chlorophyll, photosynthesis parameters, soluble proteins and ion accumulations. Exogenous PEN increased the growth parameters, pigments, photosynthesis and ion accumulations in drought stressed and unstressed plants, but the effects of PEN were more significant under water deficit conditions. PEN also reduced the negative effects of drought by osmotic balance and protein accumulations. Electrophoretic patterns indicated that PEN treatment increased the intensity of some protein bands with the molecular weights of 30 kDa in shoot and 31 kDa in roots, and several new protein bands with the molecular masses between 116 and 14 kDa appeared in leaves, shoots and roots. These results suggest that the PEN application can be a useful tool in alleviation of effects of drought stress in M. pulegium plants.     

Implications of fungal infections and mycotoxins in camel diseases in Saudi Arabia

Natural feed ingredients (corn, barley and wheat bran) and compound feed (manufactured pellet) are two types of fodder used for animal feeding, especially camel in Saudi Arabia. Twenty samples of each type of fodder were collected from seven different regions and screened for the presence of fungi, aflatoxins, ochratoxin and zearalenone. Fungal isolation of natural feed ingredients yielded 10 genera and 38 species of different fungi. Compound fodder samples were contaminated with 16 genera and 32 species of fungi. Total counts of Aspergillus, Penicillium and Fusarium in the animal feed samples were ranged from 54 to 223 × 10³, 31.9 to 60 × 10³ and 18 to 29 × 10³ CFU/g, respectively. These isolates when tested for aflatoxin, ochratoxin and zearalenone producing ability, revealed this property in only four isolate, identified as Aspergillus flavus, A. parasiticus, A. ochraceus and Fusarium graminaerum. The percentage of toxigenic fungi was ranged from 5.5% to 30% for natural feed ingredients and from 4.5% to 20% for compound feed. The incidence of aflatoxins (AFT) in samples of natural feed ingredients was found to be ranged from 1 to 24.8 ppb, ochratoxin A (OTA) ranged from 1 to 44 ppb and zearalenone (ZON) ranged from 1 to 23 ppb. Contamination of compound feed with aflatoxin and ochratoxin A was ranged from 1 to 6.4 ppb and 1 to 4.7 ppb, respectively. All samples collected were found contaminated with fungi or their toxins and natural feed samples were more contaminated compared to compound feed samples. The concentrations detected were in the allowed limit (<20 ppb) except four samples of natural feed ingredients which were above the allowed limit of the tested mycotoxins. In conclusion, feed samples were contaminated with fungi and some toxigenic isolates which were responsible about mycotoxin production. Some samples had exceeded amount of AFT, OTA and ZON and may be contaminated with other mycotoxins which mean implication of fungi in camel health problems and death in Saudi Arabia.     

Effect of ultraviolet radiation on chlorophyll,carotenoid, protein and proline contents of some annual desert plants

Investigation was carried out to find whether enhanced ultraviolet radiation influences the Malva parviflora L., Plantago major L., Rumex vesicarius L. and Sismbrium erysimoids Desf. of some annual desert plants. The seeds were grown in plastic pots equally filled with a pre-sieved normal sandy soil for 1 month. The planted pots from each species were randomly divided into equal groups (three groups). Plants of the first group exposed to white-light tubes (400–700 nm) 60 w and UV (365 nm) 8 w tubes. The second group was exposed to white-light tubes (400–700 nm) 60 w and UV (302 nm) 8 w tubes. The third group was exposed to white-light tubes (400–700 nm) 60 w and UV (254 nm) 8 w tubes, respectively, for six days. The results indicated that the chlorophyll contents were affected by enhanced UV radiation. The chlorophyll a, b, and total contents were decreased compared with the control values and reduced with the enhanced UV radiation, but the carotenoid was increased compared with the control and also reduced with the enhanced UV radiation. So, the contents of chlorophylls varied considerably. M. parviflora showed the highest constitutive levels of accumulated chlorophyll a, b, and total chlorophyll (0.463, 0.307 and 0.774 mg g⁻¹ f w) among the investigated plant species. P. major showed the lowest constitutive levels of the chloroplast pigments, 0.0036, 0.0038 and 0.0075 mg g⁻¹ f w for chlorophyll a, b, and total chlorophyll at UV-365 nm, respectively. The protein content was decreased significantly in both root and shoot systems compared with the control values but, it was increased with increasing wave lengths of UV-radiation of all tested plants. R. vesicarius showed the highest protein contents among the investigated plants; its content was 3.8 mg g⁻¹ f w at UV-365 nm in shoot system. On the other hand, decreasing ultraviolet wave length induced a highly significant increase in the level of proline in both root and shoot of all tested plants. From the results obtained, it is suggested that proline can protect cells against damage induced by ultraviolet radiation. Statistically, the variations of the studied metabolic activities were significant due to UV radiation treatment in shoot and root system of all investigated plant species.     

Mycorrhizas alter nitrogen acquisition by the terrestrial orchid Cymbidium goeringii

Background and Aims

Orchid mycorrhizas exhibit a unique type of mycorrhizal symbiosis that occurs between fungi and plants of the family Orchidaceae. In general, the roots of orchids are typically coarse compared with those of other plant species, leading to a considerably low surface area to volume ratio. As a result, orchids are often ill-adapted for direct nutrient acquisition from the soil and so mycorrhizal assocaitions are important. However, the role of the fungal partners in the acquisition of inorganic and organic N by terrestrial orchids has yet to be clarified.

Methods

Inorganic and amino acid N uptake by non-mycorrhizal and mycorrhizal Cymbidium goeringii seedlings, which were grown in pots in a greenhouse, was investigated using a ¹⁵N-labelling technique in which the tracer was injected at two different soil depths, 2·5 cm or 7·5 cm. Mycorrhizal C. goeringii seedlings were obtained by inoculation with three different mycorrhizal strains isolated from the roots of wild terrestrial orchids (two C. goeringii and one C. sinense).

Key Results

Non-mycorrhizal C. goeringii primarily took up NO₃⁻ from tracers injected at 2·5-cm soil depth, whereas C. goeringii inoculated with all three mycorrhiza primarily took up NH₄⁺ injected at the same depth. Inoculation of the mycorrhizal strain MLX102 (isolated from adult C. sinense) on C. goeringii roots only significantly increased the below-ground biomass of the C. goeringii; however, it enhanced ¹⁵NH₄⁺ uptake by C. goeringii at 2·5-cm soil depth. Compared to the uptake of tracers injected at 2·5-cm soil depth, the MLX102 fungal strain strongly enhanced glycine-N uptake by C. goeringii from tracers injected at 7·5-cm soil depth. Cymbidium goeringii inoculated with CLB113 and MLX102 fungal strains demonstrated a similar N uptake pattern to tracers injected at 2·5-cm soil depth.

Conclusions

These findings demonstrate that mycorrhizal fungi are able to switch the primary N source uptake of a terrestrial orchid, in this case C. goeringii, from NO₃⁻ to NH₄⁺. The reasons for variation in N uptake in the different soil layers may be due to possible differentiation in the mycorrhizal hyphae of the C. goeringii fungal partner.     

Transformation of pWWO in Rhizobium leguminosarum DPT to Engineer Toluene Degrading Ability for Rhizoremediation

Rhizoremediation of organic xenobiotics is based on interactions between plants and their associated micro-organisms. The present work was designed to engineer a bacterial system having toluene degradation ability along with plant growth promoting characteristics for effective rhizoremediation. pWWO harboring the genes responsible for toluene breakdown was isolated from Pseudomonas putida MTCC 979 and successfully transformed in Rhizobium DPT. This resulted in a bacterial strain (DPT^T) which had the ability to degrade toluene as well as enhance growth of host plant. The frequency of transformation was recorded 5.7 × 10⁻⁶. DPT produced IAA, siderophore, chitinase, HCN, ACC deaminase, solubilized inorganic phosphate, fixed atmospheric nitrogen and inhibited the growth of Fusarium oxysporum and Macrophomina phaseolina in vitro. During pot assay, 50 ppm toluene in soil was found to inhibit the germination of Cajanus cajan seeds. However when the seeds bacterized with toluene degrading P. putida or R. leguminosarum DPT were sown in pots, again no germination was observed. Non-bacterized as well as bacterized seeds germinated successfully in toluene free soil as control. The results forced for an alternative mode of application of bacteria for rhizoremediation purpose. Hence bacterial suspension was mixed with soil having 50 ppm of toluene. Germination index in DPT treated soil was 100% while in P. putida it was 50%. Untreated soil with toluene restricted the seeds to germinate.     

Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper)

An efficient, rapid and direct multiple shoot regeneration system amenable to Agrobacterium-mediated transformation from primary leaf with intact petiole of blackgram (Vigna mungo) is established for the first time. The effect of the explant type and its age, type and concentration of cytokinin and auxin either alone or in combination and genotype on multiple shoot regeneration efficiency and frequency was optimized. The primary leaf explants with petiole excised from 4-day-old seedlings directly developed multiple shoots (an average of 10 shoots/ explant) from the cut ends of the petiole in 95 % of the cultures on MSB (MS salts and B₅ vitamins) medium containing 1.0 μM 6-benzylaminopurine. Elongated (2–3 cm) shoots were rooted on MSB medium with 2.5 μM indole-butyric acid and resulted plantlets were hardened and established in soil, where they resumed growth and reached maturity with normal seed set. The regenerated plants were morphologically similar to seed-raised plants and required 8 weeks time from initiation of culture to establish them in soil. The regeneration competent cells present at the cut ends of petiole are fully exposed and are, thus, easily accessible to Agrobacterium, making this plant regeneration protocol amenable for the production of transgenic plants. The protocol was further successfully used to develop fertile transgenic plants of blackgram using Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron. The presence and integration of transgenes in putative T₀ plants were confirmed by polymerase chain reaction (PCR) and Southern blot hybridization, respectively. The transgenes were inherited in Mendelian fashion in T₁ progeny and a transformation frequency of 1.3 % was obtained. This protocol can be effectively used for transferring new traits in blackgram and other legumes for their quantitative and qualitative improvements.     

Peroxynitrite (ONOO?) is endogenously produced in arabidopsis peroxisomes and is overproduced under cadmium stress

Background and Aims

Peroxisomes are subcellular compartments involved in multiple cellular metabolic pathways. Peroxynitrite (ONOO⁻) is a nitric oxide-derived molecule which is a nitrating species that causes nitration of proteins. This study used cell biology techniques to explore the potential presence of peroxynitrite in peroxisomes and evaluated its content under stress conditions (excess cadmium).

Methods

Peroxynitrite, nitric oxide and superoxide anion were studied using cell-permeable specific fluorescent probes by confocal laser scanning microscopy in Arabidopsis thaliana transgenic plants expressing cyan fluorescent protein through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo.

Key Results

When no stress was applied, peroxynitrite was clearly localized in the peroxisomes of roots and stomatal guard cells. Under cadmium (150 μm) stress, the generation of peroxynitrite, nitric oxide and the superoxide anion (O₂^·–) increased and was localized in peroxisomes and the cytosol, participating in the generation of nitro-oxidative stress.

Conclusions

The results show that peroxisomes are an endogenous source of peroxynitrite, which is over-produced under cadmium stress, suggesting that the metabolism of reactive nitrogen species in peroxisomes could participate in the mechanism of the response to this heavy metal.     

Micropropagation and in vitro conservation of the rare and threatened plants Ramonda serbica and Ramonda nathaliae

Ramonda serbica and Ramonda nathaliae are rare and endemo relict plant species from Balkan Peninsula. An efficient micro propagation and in vitro conservation method via direct and indirect organogenesis from seed and leaf explants, respectively, was established in this study. The seed of both Ramonda species were collected from different populations in Kosovo, and were germinated in nutrient media JG-B without any phytohormone. The highest number of shoots and multiplication rate was observed on JG-B medium supplemented with BAP and IAA (0.5 mg l⁻¹ each), whereas the highest number of leaves per plantlets was found on WPM and RA medium supplemented with BAP and IAA (0.1 mg l⁻¹ each). During this stage of micro propagation some significant differences were observed in plantlets from different populations. The indirect organogenesis from parts of leaves of natural plants was not successful due to unavailability of established protocol for disinfections of the plant material. On other hand, parts of leaves from micro propagated plantlets, cultured on MS medium supplemented with different ratio of BAP and NAA, resulted in the highest efficiency for shoot regeneration. In vitro conservation of micro propagated plants at the lower temperature (4 °C) had a significantly positive effect for storage of more than 12 months.     

Hirsutella rhossiliensisand Verticillium chlamydosporium as Biocontrol Agents of the Root-knot Nematode Meloidogyne hapla on Lettuce

Hirsutella rhossiliensis and Verticillium chlamydosporium infected second-stage juveniles (J2) and eggs of Meloidogyne hapla, respectively, in petri dishes and in organic soil in pots planted to lettuce in the greenhouse. In vitro, H. rhossiliensis produced 78 to 124 spores/infected J2 of M. hapla. The number of J2 in roots of lettuce seedlings decreased exponentially with increasing numbers of vegetative colonies of H. rhossiliensis in the soil. At an infestation of 8 M. hapla eggs/cm³ soil, 1.9 colonies of H. rhossiliensis/cm³ soil were needed for a 50% decrease in J2 penetration of lettuce roots. Egg-mass colonization with V. chlamydosporium varied from 16% to 43% when soil was infested with 8 M. hapla eggs and treated with 5,000 or 10,000 chlamydospores of V. chlamydosporium/cm³ soil. This treatment resulted in fewer J2 entering roots of bioassay lettuce seedlings planted in the infested soils after harvesting the first lettuce plants 7 weeks after infestation with M. hapla. Hirsutella rhossiliensis (0 to 4.3 colonies/cm3 soil), V. chlamydosporium (500 to 10,000 chlamydospores/cm3 soil), or their combination, added to organic soils with 8 M. hapla eggs/cm³ soil, generally did not affect lettuce weight, root galling, or egg production of M. hapla. However, when lettuce was replanted in a mix of infested and uninfested soil (1:3 and 1:7, v:v), egg production was lower in soils with V. chlamydosporium than in soils without the fungus. Both fungi have potential to reduce the M. hapla population, but at densities below 8 eggs/cm³ soil.     

Interaction of Fusarium oxysporum f. sp. ciceri and Meloidogyne javanica on Cicer arietinum

Interaction of Meloidogyne javanica and Fusarium oxysporum f. sp. ciceri was studied on Fusarium wilt-susceptible (JG 62 and K 850) and resistant (JG 74 and Avrodhi) chickpea cultivars. In greenhouse experiments, inoculation of M. javanica juveniles prior to F. oxysporum f. sp. ciceri caused greater wilt incidence in susceptible cultivars and induced vascular discoloration in roots of resistant cultivars. Nematode reproduction was greatest (P = 0.05) at 25 °C. Number of galls and percentage of root area galled increased when the temperature was increased from 15 °C to 25 °C. Wilt incidence was greater at 20 °C than at 25 °C. Chlorosis of leaves and vascular discoloration of plants did not occur at 15 °C. The nematode enhanced the wilt incidence in wilt-susceptible cultivars only at 25 °C. Interaction between the two pathogens on shoot and root weights was significant only at 20 °C, and F. o. ciceri suppressed the nematode density at this temperature. Wilt incidence was greater in clayey (48% clay) than in loamy sand (85% sand) soils. The nematode caused greater plant damage on loamy sand than on clayey soil. Fusarium wilt resistance in Avrodhi and JG 74 was stable in the presence of M. javanica across temperatures and soil types.