Mohsen Mohamed Elsharkawy

Development of Culture Medium for the Isolation of Flavobacterium and Chryseobacterium from Rhizosphere Soil

An effective medium designated phosphate separately autoclaved Reasoner’s 2A supplemented with cycloheximide and tobramycin (PSR2A-C/T) has been developed for the isolation of Flavobacterium and Chryseobacterium strains from the plant rhizosphere. It consists of Reasoner’s 2A agar (R2A) prepared by autoclaving phosphate and agar separately and supplementing with 50 mg L−1 cycloheximide and 1 mg L−1 tobramycin. A comparison was made among the following nine media: PSR2A-C/T, PSR2A-C/T supplemented with NaCl, R2A agar, R2A agar supplemented with cycloheximide and tobramycin, 1/4-strength tryptic soy agar (TSA), 1/10-strength TSA, soil-extract agar, Schaedler anaerobe agar (SAA), and SAA supplemented with gramicidin, for the recovery of Flavobacterium and Chryseobacterium strains from the Welsh onion rhizosphere. Flavobacterium strains were only isolated on PSR2A-C/T, and the recovery rate of Chryseobacterium strains was higher from PSR2A-C/T than from the eight other media. In order to confirm the effectiveness of PSR2A-C/T, bacteria were isolated from onion rhizosphere soil with this medium. Flavobacterium and Chryseobacterium strains were successfully isolated from this sample at a similar rate to that from the Welsh onion rhizosphere.

Induction of systemic resistance against Papaya ring spot virus (PRSV) and its vector Myzus persicae by Penicillium simplicissimum GP17-2 and silica (Sio2) nanopowder

The systemic expressions of certain defense-related genes in cucumber plants induced by culture filtrate (CF) of the plant growth-promoting fungus (PGPF), Penicillium simplicissimum GP17-2, and challenging with Sio2 nanoparticles against Papaya ring spot virus (PRSV) and its vector the green peach aphid Myzus persicae were investigated. CF and Sio2 were topically applied to cucumber plants by soil drench method. Results showed that the severity of PRSV and its accumulation in cucumber leaves were significantly reduced in all treated cucumber plants compared with the control. The expression of pathogenesis-related genes showed strong activations of peroxidases (POX) and phenylalanineammonia lyase (PAL) genes and moderate expression of PR-1 one day after inoculation, while, six days after inoculation a high activation of PAL and PR-1 genes and moderate activation of POX gene were detected. No expression was detected in plants infected with PRSV alone without any treatments. These results suggested that CF and Sio2 treatments mediated the expression of the majority of various pathogen-related genes, which may contribute to the increased defense mechanism against PRSV infection in cucumber.

Control of Root Rot and Wilt Diseases of Roselle under Field Conditions

Abstract Roselle (Hibiscus sabdariffa L.) is one of the most important medicinal crops in many parts of the world. In this study, the effects of microelements, antioxidants, and bioagents on Fusarium oxysporum, F. solani, and Macrophomina phaseolina, the causal pathogens of root rot and wilt diseases in roselle, were examined under field conditions. Preliminary studies were carried out in vitro in order to select the most effective members to be used in field control trials. Our results showed that microelements (copper and manganese), antioxidants (salicylic acid, ascorbic acid, and EDTA), a fungicide (Dithane M45) and biological control agents (Trichoderma harzianum and Bacillus subtilis) were significantly reduced the linear growth of the causal pathogens. Additionally, application of the previous microelements, antioxidants, a fungicide and biological control agents significantly reduced disease incidence of root rot and wilt diseases under field conditions. Copper, salicylic acid, and T. harzianum showed the best results in this respect. In conclusion, microelements, antioxidants, and biocontrol agents could be used as alternative strategies to fungicides for controlling root rot and wilt diseases in roselle.

Mechanism of induced systemic resistance against anthracnose disease in cucumber by plant growth-promoting fungi

Plant growth-promoting fungi (PGPF) such as Phoma sp. (isolates GS8-1, GS8-2 and GS8-3) and non-sporulating fungus (isolate GU21-2) were tested for their ability to induce systemic resistance against Colletotrichum orbiculare in cucumber. These isolates, used as colonized barley kernels to natural soils, induced systemic resistance in the greenhouse as well as in the field. Different elicitors from PGPF such as cell walls retaining or lacking protein and lipids, cell walls lipid fraction, and three fractions of culture filtrate (CF) with different molecular weight ranges were tested for their ability to elicit the defense response of cucumber plants under controlled conditions. Cell wall fraction lacking protein and lipids and the cell wall lipid fraction from root colonizing isolates GS8-1, GS8-2, and GS8-3 protected plants against C. orbiculare. While, only cell wall lipid fraction and CF fractions of different molecular weight ranges of the isolate GU21-2 protected plants against C. orbiculare infection. Methanol-soluble substances from CF fractions with molecular weight greater than 12,000 and less than 8000 consistently protected plants against the pathogen. Germination of C. orbiculare spores decreased significantly at 72 h of incubation on leaves of PGPF-protected plants. Isolate GU21-2 was the most effective in inhibiting spore germination. On the other hand, isolates GS8-1 and GU21-2 induced lignifications in the hypocotyls of seven-day-old cucumber seedlings after challenge inoculation with C. orbiculare. Three-week-old cucumber plants treated with isolate GU21-2 and challenged with C. orbiculare showed increased activities of exo- and endo-forms of glucanase and chitinase, as well as peroxidase and polyphenol oxidase in the second true leaves. Induction treatment with isolate GS8-1 also increased the activities of these enzymes with the exception of exo-glucanase. This study shows that the inoculation of PGPF or its CF resulted in additive effect on the suppression of anthracnose disease in cucumber.

Control of Bean common mosaic virus by plant extracts in bean plants

The present study aimed to evaluate some plant extracts from Plectranthus tenuiflorus, Azadirachta indica, Clerodendrum inerme, Schinus terebinthifolius and Mirabilis jalapa as antiviral materials against Bean common mosaic virus (BCMV) in bean plants. The results showed that all the tested plant extracts were effective in reducing the number of local lesions formed by BCMV. The mixing of P. tenuiflorus extracts with BCMV inoculum achieved the highest reduction in BCMV infection (92%), while the mixing of S. terebinthifolius extracts with BCMV inoculum recorded the lowest reduction in BCMV infection (68%). Seed treatment with 1% (w/v) of P. tenuiflorus showed the highest reduction in disease incidence (93% and 85%) under greenhouse and field conditions, respectively. Spray treatments of P. tenuiflorus and M. jalapa reduced disease incidence to 12% and 17% under greenhouse conditions, whereas under field conditions they reduced disease incidence to 17% and 23%, respectively. Protein analysis revealed different effects of the tested treatments on bean plants indicating different gene(s) expression. This study suggests the possibility of using plant extracts for controlling BCMV infection.

A nonpathogenic species of binucleate Rhizoctonia inhibits the formation of infection structures caused by Rhizoctonia solani on cucumber

Binucleate Rhizoctonia (BNR) W7 was evaluated for the efficacy to control damping-off disease in cucumber seedlings. Results revealed that Lobate appresoria and dome-shaped infection cushion were produced by virulent Rhizoctonia solani C9 AG4 on the surface of the cucumber seedling hypocotyls, causing damping-off, 12 h after inoculation. A significant positive correlation was observed between disease severity rating and number of infection cushions (r = 0.94) and appresoria (r = 0.97) on the hypocotyls of seedlings inoculated with virulent R. solani C9 alone. Inoculation of a nonpathogenic species of BNR prior to virulent R. solani significantly reduced the number of appresoria on hypocotyls surface and inhibited the formation of infection cushions that accompanied by highly significant (P ≤ 0.01) reduction in disease severity in comparison with seedling inoculated with R. solani C9 alone. Formation of mucilaginous material upon recognition of the nonpathogenic isolate of BNR followed by hyphal lysis of BNR W7 6 h after inoculation was responsible for reduction in appresorium formation and complete inhibition of infection cushion formation. However, the number of infection structures produced on cucumber was not significantly different for hypocotyls of seedlings inoculated with R. solani C9 with and without the nonpathogenic BNR W7.

Integrated control of rice kernel smut disease using plant extracts and salicylic acid

The effects of salicylic acid (SA) at three concentrations i.e. 2.5, 5 and 7 mM and plant extracts from pick tooth (Ammi visnaga), liquorice (Glycyrrhiza glabra), artemisia (Artemisia judaica), mint (Mentha viridis), clove (Syzygium aromaticum) and blue gum (Eucalyptus globulus) on the infection of rice kernel smut disease caused by Tilletia barclayana were studied. Spraying of rice plants with different concentrations of SA at seven days before infection was the most effective treatment against pathogen infection. Among all plant extract treatments, M. viridis and S. aromaticum were the most effective treatments. Additionally, our results showed increased levels of peroxidase, polyphenol oxidase, phenylalanine ammonia lyase and chitinase as well as total protein contents in the treated plants compared with the control. In conclusion, accumulations of these oxidative enzymes in plants treated with SA and plant extracts provide their role in the activation of induced resistance against T. barclayana.

Control of tomato bacterial wilt and root-knot diseases by Bacillus thuringiensis CR-371 and Streptomyces avermectinius NBRC14893

Ralstonia solanacearum and Meloidogyne incognita are two soilborne pathogens that cause serious damage and great losses in the production of tomato. For this purpose, a bacterial isolate, Bacillus thuringiensis CR-371, and an actinomyces isolate, Streptomyces avermectinius NBRC14893, were examined for their ability to protect tomato from root-knot nematode and bacterial wilt diseases under glasshouse conditions. Treatment of tomato roots with B. thuringiensis CR-371 and S. avermectinius NBRC14893 followed by challenge inoculation with R. solanacearum and M. incognita significantly decreased disease severity of bacterial wilt alone, root-knot nematode alone, or mixed infection by both pathogens compared to the control. Furthermore, pretreatment of tomato roots with B. thuringiensis CR-371 and S. avermectinius NBRC14893 significantly reduced bacterial proliferation of R. solanacearum both in pathogen alone inoculated plants and in plants co-inoculated with R. solanacearum and M. incognita. In conclusion, our results suggest that the treatment of tomato roots with B. thuringiensis CR-371 and S. avermectinius NBRC14893 simultaneously suppresses bacterial wilt and root-knot nematode diseases. Therefore, B. thuringiensis CR-371 and S. avermectinius NBRC14893 could provide new options for integrated pest management strategies against plant diseases, especially against bacterial-nematode disease complexes that cause synergistic yield losses.

Characterization of 16SrII-D subgroup associated phytoplasmas in new host plants in Egypt

Abstract Samples of three plant species displaying phytoplasma symptoms were collected from Kafrelsheikh and Al-Gharbia governorates during 2014. Witches’ broom and virescence symptoms were observed in periwinkle (Catharanthus roseus). Onion (Allium cepa) plants showed yellowing, streaks and twisting and Opuntia abjecta with proliferation and cylindrical of cladodes. Total DNA was extracted from symptomatic and asymptomatic plants, and phytoplasma were detected in all 12 symptomatic plants collected through direct and nested PCR assays with primers P1/P7 and R16F2n/R16R2. The results of phylogenetic analysis revealed that the phytoplasma isolates belong to 16SrII group. With a nucleotide identity greater than 98.7% with three members of 16SrII group, Papaya yellow crinkle, Y10097; “Ca. P. aurantifolia”, U15442; and peanut witches’ broom, Al33765, the strains identified in this study are “Ca. P. aurantifolia”-related strains. Virtual RFLP analysis of the 16S rRNA gene sequences with 17 restriction enzymes confirmed that the phytoplasma isolates belong to the “Candidatus Phytoplasma australasia” 16SrII-D subgroup. To the best of our knowledge, periwinkle, onion and Opuntia abjecta are considered new hosts for 16SrII group in Egypt.

Suppression of rice blast, cabbage black leaf spot, and tomato bacterial wilt diseases by Meyerozyma guilliermondii TA-2 and the nature of protection

This study was conducted to evaluate the efficacy of yeast strain TA-2 for controlling rice blast, cabbage black leaf spot, and tomato bacterial wilt diseases. Microscopic and phylogenetic analyses based on rDNA-internal transcribed region (ITS) and rDNA-D1/D2 sequences indicated that yeast strain TA-2 is Meyerozyma guilliermondii. Pretreatment with TA-2 by soil drenching significantly reduced the severity of black leaf spot disease caused by Alternaria brassicicola and leaf blast disease caused by Magnaporthe oryzae. Symptom development of tomato bacterial wilt caused by Ralstonia solanacearum in both soil drench and needle inoculation tests was significantly reduced in TA-2-pretreated plants under soil drenching. Disease severity and R. solanacearum growth were significantly reduced in tomato plants pretreated with yeast culture, cell suspension, or culture filtrate of TA-2 under soil drenching. TA-2 does not produce antibiotics. The present study indicates that disease suppression is systemic, as the roots were treated with TA-2 and the pathogens were inoculated onto leaves or stems, thereby separating the two spatially. M. guilliermondii TA-2 could become a promising natural antimicrobial agent against rice blast, cabbage black leaf spot, and tomato bacterial wilt diseases and might be useful as an eco-friendly control measure, contributing to sustainable agriculture.