Rhizopus Stem Rot of Orobanche aegyptiaca caused by Rhizopus oryzae in China

Stem rot was recorded on Orobanche aegyptiaca in Shihezi City, Xinjiang Uygur Autonomous Region, China from 2010 to 2011. The pathogen was isolated repeatedly from the infected stems and was identified as Rhizopus oryzae based on morphology, cultural features and molecular analysis. Koch's postulates were supported by pathogenicity tests conducted on healthy plants grown on processing tomato and melon. To our knowledge, this paper is the first to report the occurrence of R. oryzae stem rot on O. aegyptiaca.     

Carvacrol and eugenol effectively inhibit Rhizopus stolonifer and control postharvest soft rot decay in peaches

Aims

This study aimed to investigate the antifungal mechanism of carvacrol and eugenol to inhibit Rhizopus stolonifer and the control of postharvest soft rot decay in peaches.

Methods and Results

To investigate the antifungal mechanism, the effects of carvacrol and eugenol on the mycelium growth, leakages of cytoplasmic contents, mycelium morphology, cell membrane and membrane composition of R. stolonifer were studied. Carvacrol and eugenol both exhibited dose‐dependent antifungal activity against R. stolonifer, carvacrol at a concentration of 2 μl per plant and eugenol at a concentration of 4 μl per plant inhibited fungal growth completely. The two essential oils (EOs) increased cell membrane penetrability and caused the leakage of cytoplasm, nucleic acid and protein content. The observation using scanning electron microscopy and fluorescent microscopy showed modification of the hyphal morphology and breakage of the cell plasma membrane. Decreased ergosterol contents confirmed that the two EOs could destroy the membrane of R. stolonifer. For the in vivo test, the inhibition of soft rot disease and the induction of defence‐related enzymes were investigated. Carvacrol and eugenol significantly reduced the incidence and severity of soft rot decay in inoculated peaches. The best treatments for controlling soft rot decay were obtained at 0·5 μl l^?1 for carvacrol and 1 μl l^?1 for eugenol. The activities of defence‐related enzymes in peaches were also enhanced by fumigation with two EOs.

Conclusion

This study showed that carvacrol and eugenol could effectively inhibit the growth of R. stolonifer in vitro and successfully control the incidence of soft rot decay in honey peaches.

Significance and Impact of the Study

The above findings may be the main antifungal mechanism of carvacrol and eugenol on R. stolonifer. Furthermore, carvacrol and eugenol are helpful for their commercial application on the preservation of fresh fruit.     

Morphological and Physiological Changes on Rhizopus stolonifer by Effect of Chitosan,Oligochitosan or Essential Oils

Effects of chitosan, oligochitosan and the essential oils of clove and cinnamon were evaluated on hyphal morphology, cell wall thickness, minimum medium pH changes and respiration of Rhizopus stolonifer. Changes in hyphal morphology were observed due to chitosan or oligochitosan treatment in this fungus. Mycelial branching, abnormal shapes and swelling were showed on hyphae of R. stolonifer treated with chitosan, whereas the development of hyphae was markedly inhibited by the effect of oligochitosan. Clove and cinnamon oils caused few morphological changes in the hyphae of R. stolonifer. Cell wall thickness was increased approximately 2‐ to 3‐fold by effect of chitosan, oligochitosan and the essential oil of clove. R. stolonifer grown in minimum medium generated a decrease in the medium's pH. However, the addition of chitosan or oligochitosan caused increases in pH of medium culture. The highest pH value (5.4) was observed in the presence of chitosan. The respiration of R. stolonifer was stimulated at low concentrations of chitosan, oligochitosan or essential oils. Significant changes in morphology and physiology of this fungus were demonstrated by the effect of all evaluated compounds. The most important changes were induced on cells of R. stolonifer treated with chitosan and oligochitosan.     

Control of storage rot of cassava tuber caused by Rhizopus oryzae using some plant extracts

Abstract

The potency of Piper nigrum seed and leaf, Aframomum meleguata seed and Ageratum conyzoides leaf extracts in the control of cassava tuber rot caused by Rhizopus oryzae was investigated. Water, ethanol and petroleum ether were used as extracting solvents. These extracts were fungitoxic both in vitro and in vivo against the test pathogen. P. nigrum seed extracts were the best followed by those of A. meleguata seed, A. conyzoides leaf and then P. nigrum leaf. The extracts were more effective in controlling rot development in unwounded than wounded tubers especially when they were applied before inoculation with the test pathogen. Ethanol extracts gave the highest growth inhibition in vivo followed by water and the petroleum ether extract whereas water extracts showed marked superiority over the extracting solvents in checking rot development in cassava tuber. Water and ethanol extracts of P. nigrum seed and leaf, A. meleguata seed and A. conyzoides leaf could be used as pesticide of plant origin in the control of R. oryzae causing cassava tuber rot in storage.     

Identification and Characterization of Rhizot,a Novel LTR Retrotransposon of Rhizopus oryzae and R. delemar

A novel retrotransposon Rhizot was identified in Rhizopus oryzae and R. delemar. Rhizot has a unique structure that consists of a pol ORF similar to non-LTR (long terminal repeat) retorotransposons between two LTRs. Rhizot was distributed in all Rhizopus species tested. The Rhizot pol gene was transcribed in the liquid culture, and was induced by UV and oxidative stress.     

Comparison of Sucrose-Hydrolyzing Enzymes Produced by Rhizopus oryzae and Amylomyces rouxii

Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.     

Effects of iron and desferrioxamine on Rhizopus infection

To investigate the association among iron, desferrioxamine, and a Rhizopus infection, the influence of iron and/or desferrioxamine on experimental mucormycosis in mice was examined. All mice pretreated with iron, desferrioxamine, or a combination of iron and desferrioxamine died within 5 days after the inoculation of R. oryzae. In the mice fungal lesions were observed in the brain which resembled human cerebral mucormycosis. By contrast, the mortality in the control mice with R. oryzae was 20% through the 3-week experimental period. Therefore, it was demonstrated that iron as well as desferrioxamine administration markedly promotes the growth of R. oryzae. The increased susceptibility to R. oryzae was considered to be due to increased serum iron in the animals pretreated with iron only; however, pretreatment with desferrioxamine did not affect the amount of serum ion. Thus, the data suggest that desferrioxamine acts as a siderophore to R. oryzae and exerts an adverse effect on mucormycosis. This study has shown that the presence of iron and desferrioxamine enhances the virulence and pathogenicity of R. oryzae by serving as a growth factor.     

Preparation and testing of Sardinella protein hydrolysates as nitrogen source for extracellular lipase production by Rhizopus oryzae

Various fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were used as nitrogen sources for the production of extracellular lipase by the filamentous fungus Rhizopus oryzae. The best results were obtained with defatted meat–fish protein hydrolysates (DMFPH), indicating the presence in the lipid fraction of some constituents which may repress lipase synthesis. Furthermore, it was found that the extensive hydrolysis of fish proteins resulted in a higher lipase production. The use of 40 g DMFPH l^–1 for the growth of Rhizopus oryzae in medium R1 resulted in a lipase production of 394 U ml^–1, higher than the yield obtained with standard soy peptone as nitrogen source (373 U ml^–1). The most appropriate medium for the growth and the production of lipase is composed only of 24 g DMFPH l^–1 and 10 g glucose l^–1, indicating that the strain can obtain its nitrogen and salts requirements directly from fish substrate.     

Evaluation of Clove Essential Oil as a Mycobiocide Against Rhizopus stolonifer and Fusarium solani,Tuber Rot Causing Fungi in Yam (Dioscorea rotundata Poir.)

To exploit natural products for plant disease control, the essential oil of Syzygium aromaticum (L.) Merr. & Perr. (clove) was investigated for its antifungal activity against Rhizopus stolonifer and Fusarium solani, the postharvested yam tuber rot pathogens. The essential oil was obtained by hydrodistillation using a Clevenger‐type apparatus. The chemical composition of the oil was determined by gas chromatography and gas chromatography coupled with mass spectrometry. Antifungal activities of the oil were tested in vitro against mycelia growth and spores germination. In situ tests were conducted on healthy yam tubers, and necrosis symptoms were assessed. Results showed that eugenol (79.4%), eugenylacetate (9.2%) and isocaryophyllene (7.0%) were the major components. The oil exerted antifungal activities with total inhibition (TI) of the mycelia growth of R. stolonifer and F. solani was recorded at 200 and 300 ppm, respectively, while TI of spores germination was recorded at 31.25 and 250 ppm, respectively. For the standard fungicide (Ridomil^®), TI value of mycelia growth was 1600 ppm for the both pathogens, while TI of spores germination were 200 ppm and 1600 ppm, respectively, for Rhizopus and Fusarium. In situ tests showed complete inhibition of yam tuber rot when the essential oil was applied at 2000 ppm for preventive tests. This oil also reduced significantly (P ≤ 0.05) necrosis development on yam tuber for curative test at the same concentration. Total inhibition of the necrosis by Ridomil (3000 ppm) was observed only for Rhizopus on preventive test. There were positive correlations between the oil concentration and the reduction of necrosis cause by R. stolonifer and F. solani. These findings showed that clove essential oil may serve as environmental friendly bio‐fungicide for the management of postharvest yam tuber rot.     

Purification of the extracellular pectinolytic enzyme from the fungus Rhizopus oryzae NBRC 4707

The pectinolytic enzyme from the solid-state culture of Rhizopus oryzae NBRC 4707 was purified to homogeneity by column chromatography on CM-Toyopearl 650 M and hydroxylapatite. The molecular weight of the enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 31,000 and was reduced to 29,700 after treatment with endoglycosidase H. Maximal activity was observed near pH 4.5 at 45°C. The enzyme was shown to be endopolygalacturonase, as judged from the formation of oligogalacturonides as its reaction products. The addition of purified enzyme, as expected, enhanced the formation of lactic acid and ethanol in potato pulp grown with R. oryzae.     

A flax-retting endopolygalacturonase-encoding gene from Rhizopus oryzae

A polygalacturonase from the filamentous fungus Rhizopus oryzae strain sb (NRRL 29086), previously shown to be effective in the retting of flax fibers, was shown by the analysis of its reaction products on polygalacturonic acid to be an endo-type. By zymogram analysis, the enzyme in the crude culture filtrate appeared as two active species of 37 and 40 kD. The endopolygalacturonase-encoding gene was cloned in Escherichia coli and its translated 383-amino acid sequence found to be identical to that of a presumed exopolygalacturonase found in R. oryzae strain YM9901 and 96% identical to a hypothetical protein (RO3G_04731.1) in the sequenced genome of R. oryzae strain 99–880. Phylogenetic analysis revealed the presence of an unique cluster of Rhizopus polygalacturonase sequences that are separate from other fungal polygalacturonases. Conservation of 12 cysteines appears to be a special feature of this family of Rhizopus polygalacturonase sequences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Pantoea stewartii Causing Stewart's Wilt on Dracaena sanderiana in Korea

In April 2010, a severe occurrence of Stewart's wilt on Dracaena sanderiana plants was observed in greenhouses in Seongnam, Gyeonggi Province, South Korea, with an incidence of 35‐50%. Being imported plants, little was known about the pathogens associated with D. sanderiana. Symptoms included chlorosis, wilting and leaf blight on the leaf surfaces. Physiological analysis, pathogenicity tests, sequencing and phylogenetic analysis of the 16S rRNA gene revealed that the pathogen was the bacterium Pantoea stewartii. To the best of our knowledge, this is the first report on bacterial wilt caused by P. stewartii on D. sanderiana.     

Bacterial iturins mediate biocontrol activity of Bacillus sp. against postharvest pear fruit‐rotting fungi

A potential antagonist, Bacillus sp. LYLB4 isolated from pear fruits, was tested for its antifungal activity against postharvest pear pathogens. LYLB4 had a remarkable antifungal effect on Botryosphaeria dothidea. Although it showed a weak inhibition effect on the growth of Rhizopus stolonifer on potato dextrose agar (PDA) plates, LYLB4 almost completely destroyed R. stolonifer during direct contact in potato dextrose broth (PDB). LYLB4 treatment was able to significantly reduce disease incidence (by 68.9% and 100%, respectively) and lesion diameter (by 68.7% and 100%, respectively) of ring rot caused by B. dothidea and soft rot caused by R. stolonifer in pears. LYLB4 also suppressed several other phytopathogens in vitro, suggesting a broad‐spectrum antagonistic activity against fungal pathogens. 16S rRNA and gyrA sequence analysis indicated that LYLB4 is closely related to B. velezensis. Genome mining indicated that LYLB4 had 11 secondary metabolites encoding clusters, but that the surfactin and fengycin gene clusters may not be functional because of a large deletion. Matrix‐assisted laser desorption ionization‐time of flight mass spectra (MALDI‐TOF‐MS) demonstrated that iturins were the major lipopeptides, and that C₁₆/C₁₇ Bacillomycin D synthesis was stimulated when LYLB4 was co‐cultured with B. dothidea compared to the control. Overall, these results demonstrate that the main biocontrol mechanism adopted by LYLB4 could be through the production of toxic metabolites and direct contact with pathogens.     

Histopathological and biochemical aspects of grafted and non-grafted cucumber infected with stem rot caused by Fusarium spp.

Cucumber grafting has been used in Egypt recently to induce soil diseases tolerance. The impact of various grafting techniques on the vulnerability of grafted cucumber seedlings to Fusarium which stimulates the stem rot was investigated. Consequently, the anatomical and physiological studies were carried out on the diseased and healthy grafted cucumber seedlings, comparing with the non-grafted ones. Fusarium equiseti (MW216971.1) caused a severe stem rot of the grafted seedling through affecting the connection area of the different grafting methods, leading to complete seedling death. The hole insertion grafting method significantly exhibited the highest diseases incidence (100%), and mean disease severity index (5) when inoculated with F. equiseti. The pathogen remarkably affected the graft union area causing tissue discoloration and decay. The levels of antioxidant enzymes and total phenols were significantly enhanced in the diseased grafted and self-rooted cucumber. However, the diseased grafted cucumber recorded significantly the highest values of the antioxidant enzymes activities and total phenolic content when compared with the self-rooted ones. The results of SDS-PAGE profile revealed variations in the leaves protein profile of the grafted and self- rooted seedlings in response to Fusarium infection. Taken together, grafting cucumber onto a resistant rootstock using the splice technique can alleviate the stem rot severity caused by Fusarium spp. by enhancing the histological, physiological and molecular defense response of the grafted seedling.     

Genetic diversity in <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> strains as revealed by the sequence of lactate dehydrogenase genes

Twenty-seven strains of Rhizopus oryzae accumulating predominantly lactic acid were shown to possess two ldh genes, ldhA and ldhB, encoding NAD-dependent lactate dehydrogenases. Variation in nucleotide sequence was identified for each gene from different strains, and similar phylogenetic trees were obtained based on the nucleotide sequences of both genes. The other 21 strains of R. oryzae accumulating predominantly fumaric and malic acids contained a single ORF of ldhB. Compared to the strains accumulating predominantly lactic acid, a lower degree of sequence divergence was found in ldhB, resulting in a separate cluster in the phylogenetic tree. The high similarity (>90%) spanning the ORF and adjacent regions demonstrates that ldhA and ldhB are derived from the same ancestor gene. The strains accumulating predominantly fumaric and malic acids lack functional ldhA, which plays a role in lactic acid synthesis and may form a lineage separated from the strains accumulating predominantly lactic acid in the genus Rhizopus.     

The active component in the flax-retting system of the zygomycete Rhizopus oryzae sb is a family 28 polygalacturonase

The zygomycete Rhizopus oryzae sb is a very efficient organism for retting of flax, the initial microbiological step in the process of making linen. An extracellular polygalacturonase, when isolated could perform retting, and therefore probably is the key component in the retting system of R. oryzae. This was purified and characterized. The purified enzyme has a molecular mass of 37,436 Da from mass spectrometric determination, an isoelectric point of 8.4, and has non-methylated polygalacturonic acid as its preferred substrate. Peptide sequences indicate that the enzyme belongs to family 28, in similarity with other polygalacturonases (EC. 3.2.1.15). It contains, however an N-terminal sequence absent in other fungal pectinases, but present in an enzyme from the phytopathogenic bacterium Ralstonia solanacearum. The biochemical background for the superior retting efficiency of R. oryzae sb is discussed.     

Production and characterization of chitosan obtained from <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> grown on potato chip processing waste

Potato chip processing waste of trimmed potato, potato peel and substandard (low-quality) potato chips, obtained from a potato chip processing plant, were used as substrates for chitosan production from Rhizopus oryzae. It was cultured on each waste product at 30 ± 2°C and 70% moisture content for 21 days. Fermented potato peel had the highest yield after 5 days of fermentation. The cultivation condition of chitosan obtained from R. oryzae was optimum for a peel size of less than 6 mesh, 70% moisture content and a pH of 5. Furthermore, the best extraction condition was using 46% sodium hydroxide at 46°C for 13 h followed by 2% acetic acid at 95°C for 8 h. The maximum chitosan yield obtained by these conditions was 10.8 g/kg substrate. Fungal chitosan properties were found to be 86–90% degree of deacetylation, molecular weight of 80–128 kDa and viscosity of 3.1–6.1 mPa s. Therefore, potato peel could be applied as a low cost substrate for chitosan production from R. oryzae.     

An in vitro investigation of Malaysian Phytophthora palmivora isolates and pathogenicity study on oil palm

Bud rot disease affecting oil palm in South American countries is reported to be caused by Phytophthora palmivora. P. palmivora is a local pathogen affecting various crops in Malaysia, and this finding caused an alarm, which prompted an investigation of pathogenicity using Malaysian P. palmivora to assess the potentials of this Oomycete to infect oil palm in Malaysia. A total of 11 P. palmivora isolates were obtained from cocoa and durian for the study. Leaf bioassays via artificial inoculation using 50,000 zoospores/ml and mycelial agar disc showed severe necrotic lesions on the infection spot of oil palm (DxP) spear leaves. Mild infection was observed in oil palm spear leaves of OxG hybrids indicating lower susceptibility against P. palmivora infection. Phylogenetic analysis using internal transcribed spacer (ITS) data revealed that Malaysian isolates were genetically similar to Colombian isolates supported by significant bootstrap values. The leaf bioassay results revealed that Malaysian oil palm materials are susceptible towards local P. palmivora infection. The Colombian P. palmivora isolates causing bud rot incidence may have evolved over a long period of time, undergone sequential genetic shift to become more virulent towards Colombian oil palm planting materials.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.