Solid-state fermentation with Pleurotus ostreatus improves the nutritive value of corn stover-kudzu biomass.

A batch culture technique was used to evaluate dry matter (DM) digestibility of corn stover (Zea mays L.) and kudzu, Pueraria montana (Lour.) Merr. after solid-state fermentation (SSF) with a white-rot fungus, Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm. Five dietary treatments consisting of mixtures of corn stover (C) and kudzu (K) in varying ratios, (1) 100C:0K, (2) 75C:25K, (3) 50C:50K, (4) 25C:75K, and (5) 0C:100K, were inoculated with P. ostreatus (MBFBL 400) and subjected to SSF for 0, 35, and 77 days. The study was arranged as a 5 × 3 factorial design with 3 replicates. Grass hay was included in the study as a control. Interactions (P < 0.05) between treatments and fermentation time were noted for the fermentation kinetics. Asymptotic gas was the highest (P < 0.05) for 0C:100K and 100C:0K on day 77. Treatment effect (P < 0.001) and treatment × fermentation time interaction (P < 0.001) were noted for in vitro dry matter digestibility (IVDMD). On day 77, treatment 4 had the highest (P < 0.001) IVDMD value, while treatment 1 had the lowest (P < 0.001) IVDMD. There was no difference (P > 0.05) between treatments 3, 5, and control. Numerically, the ranking of their IVDMD values from the highest to the lowest is 4 > 2 > 5 > control >3 > 1. The results show that the treated corn stover and kudzu mixes were comparable with the control, which is good quality hay. This is the first report that demonstrates the potential use of a combined mixture of corn stover and kudzu in ruminant animal feed development.

A Nodulation-Proficient Nonrhizobial Inhabitant of Pueraria phaseoloides.

Pueraria phaseoloides is a legume cover crop, found chiefly in the wet zone of Sri Lanka. Nitrogen fixation is performed by nodular inhabitants of this cover crop, comparable to the nodule-dwelling bacteria of most other legume plants. We isolated a bacterium (Sub1) from Pueraria phaseoloides, of coccobacillus cell shape, that showed nodulation, when assessed by hydroponics, showing nodules as early as 3 weeks after reinfection. When a nifH fragment from the genome of this bacterium was amplified using a pair of nifH primers, it yielded an amplicon of 360 bp that, when sequenced, helped us identify the bacterium, as belonging to a species of Pseudacidovorax intermedius, at 99% sequence identity. When we constructed a phylogenetic tree with neighboring sequences, we encountered nifH sequences of Pseudacidovorax, forming a monophyletic cluster, which too contained a single Azospirillum species. The genus Pseudacidovorax is a bacterium that, so far, has not been associated with legume nodules. Sub1 secreted a pair of enzymes to the extracellular medium to degrade cellulose and milk proteins. The Sub1 bacterium showed biofilm formation and secreted into the extracellular medium, indole acetic acid. Sub1 also showed a "bulls eye" swarming pattern for the chemoattractant proline, while showing no significant chemotaxis movement, for naringenin, quercetin, and glutamate. Sub1 too possesses the basic genetic foundation (nifH and nifD) to produce a molybdenum-dependent nitrogenase enzyme. We finally show that this rare nonrhizobial bacterium is able to impact, positively, nodulation and shoot length of Pueraria plants, demonstrating that this beta-proteobacterium can abet the biological vigor of this legume cover crop.

Flavonoid-rich plants used as sole substrate to induce the solid-state fermentation of laccase.

High cost becomes the major obstacle for the industrial application of laccase. Many approaches have been applied to enhance the yield and decrease the cost of laccase. Since flavonoids are the natural inducers for laccase production, in this article, flavonoid-rich plants were taken as the sole substrate for the solid-state fermentation of Funalia trogii (Cui 3676). It indicated that flavonoid-rich plants can effectively promote the production of F. trogii laccase without the addition of inducers. The laccase activity was 42.5 IU g(-1) substrate when kudzu vine root was used as the substrate, which was enhanced by 4.46 times than that when bran was used as the substrate. Meanwhile, the solid-state fermentation of laccase could enrich flavonoids, benefiting their extraction. The content of flavonoids extracted from fermented kudzu vine root and Ginkgo biloba leaves was enhanced by 56.41 and 24.11 %, respectively, compared to the unfermented substrate, and the relative reductive ability and scavenging ability of hydroxyl radicals of flavonoids in the fermented residues were essentially unchanged. Thus, flavonoid-rich plants will become a kind of potential substrate for laccase fermentation which is beneficial in enhancing the yield and reducing the cost of laccase.

Ovularia puerariae Sawada is the hyphomycetous anamorph of a new Marasmius species on living leaves of kudzu (Pueraria montana, Fabaceae).

An arthroconidial hyphomycete on living leaves of kudzu (Pueraria montana, Fabaceae), originally described by Sawada in 1959 as Ovularia puerariae, was rediscovered. This anamorph is connected to an unknown Marasmius teleomorph belonging to section Globulares, which develops on the same living leaves. Ultrastructure and LSU rDNA sequence analysis of the anamorph confirm this connection. The fungus does not have only a unique biology among agarics, comparable only to Mycena citricolor, but also has the potential for application as a control agent of kudzu. During comparison with similar anamorph genera, Illosporium graminicola was found to be a synonym for Beniowskia sphaeroidea.

Bioherbicidal activity from washed spores of Myrothecium verrucaria.

The fungal plant pathogen, Myrothecium verrucaria, is highly virulent to several important weed species and has potential utility as a bioherbicide. However the production of macrocyclic trichothecene mycotoxins by this fungus presents significant safety concerns. It was discovered that trichothecenes are removed from M. verrucaria spores by repeated washes with water. These washed spores retained bioherbicidal efficacy against kudzu when tested in field trials and on sicklepod when tested under greenhouse conditions. Changes in the growth medium combined with washing spores with water resulted in greater than 95% reduction in roridin A and verrucarin A. Washing spores reduced trichothecene concentrations in spore preparations with no significant effect on plant biomass reduction, thus demonstrating the possibility of M. verrucaria formulations with improved safety to researchers, producers and applicators.

Mechanism of acid tolerance in a rhizobium strain isolated from Pueraria lobata (Willd.) Ohwi.

The Rhizobium sp. strain PR389 was isolated from the root nodules of Pueraria lobata (Willd.) Ohwi, which grows in acidic (pH 4.6) yellow soil of the Jinyun Mountains of Beibei, Chongqing, China. While rhizobia generally have a pH range of 6.5-7.5 for optimum growth, strain PR389 grew in a liquid yeast extract - mannitol agar medium at pH 4.6, as well as in a pH 4.1 soil suspension, suggesting acid tolerance in this specific strain of rhizobium . However, at pH 4.6, the lag phase before vigorous growth was 40 h compared with 4 h under neutral conditions (pH 7.0). For PR389, the generation time after the lag phase remained the same at different pH levels despite the different durations of the lag phase. Except in the pH 4.4 treatment, the pH of the culturing media increased from 4.6, 4.8, 5.0, and 5.5 to neutral and slightly alkaline after 70 h of culture. Chloramphenicol was added to determine if protein production was involved in the increasing pH process. Chloramphenicol significantly inhibited PR389 growth under acid stress but had little effect under neutral conditions. Proton flux measured during a short acid shock (pH 3.8) revealed that this strain has an intrinsic ability to prevent H(+) from entering cells when compared with acid-sensitive rhizobia. We propose that the mechanism for acid tolerance in PR389 involves both intracellular and extracellular processes. When the extracellular pH is lower than pH 4.4, the cell membrane blocks hydrogen from entering the cell. When the pH exceeds 4.4, the rhizobium strain has the ability to raise the extracellular pH, thereby, potentially decreasing the toxicity of aluminum in acid soil.

Characterization of kudzu (Pueraria spp.) resistance to Phakopsora pachyrhizi, the causal agent of soybean rust.

Kudzu (Pueraria spp.) is an accessory host for soybean rust (SBR) (caused by Phakopsora pachyrhizi) that is widespread throughout the southeastern United States. An expanded survey of kudzu sites was conducted in 2008 to determine the proportion of natural resistance in the north-Florida kudzu population. Of the 139 sites evaluated, approximately 18% were found to be free of SBR infection, while 23% had reduced sporulation. Ten accessions of kudzu from north-central Florida were characterized for their response to challenge by a single isolate of P. pachyrhizi under laboratory conditions. Three outcomes were observed: tan lesions with profuse sporulation (susceptible); reddish-brown lesions with delayed, reduced sporulation (resistant); and an immune response in which no lesions developed (immune). Of the 10 accessions, 6 were susceptible, 3 were immune, and 1 was resistant. Cytological examination revealed that resistant interactions were typified by early onset of a multicell hypersensitive response (HR) while typical immune interactions were the result of cell wall depositions that blocked penetration in combination with early onset of the HR. Quantitative real-time polymerase chain reaction was performed to determine the extent of colonization. After 15 days, there was 10-fold less P. pachyrhizi DNA present in resistant compared with susceptible kudzu, while the amount of P. pachyrhizi DNA present in the immune kudzu was below the detection level. Susceptible kudzu had approximately half the amount of P. pachyrhizi DNA present when compared with a susceptible soybean cultivar.

Modeling landscape-scale pathogen spillover between domesticated and wild hosts: Asian soybean rust and kudzu.

Many emerging pathogens infect both domesticated and wild host species, creating the potential for pathogen transmission between domesticated and wild populations. This common situation raises the question of whether managing negative impacts of disease on a focal host population (whether domesticated, endangered, or pest) requires management of only the domesticated host, only the wild host, or both. To evaluate the roles of domesticated and wild hosts in the dynamics of shared pathogens, we developed a spatially implicit model of a pathogen transmitted by airborne spores between two host species restricted to two different landscape patch types. As well as exploring the general dynamics and implications of the model, we fully parameterized our model for Asian soybean rust, a multihost infectious disease that emerged in the United States in 2004. The rust fungus Phakopsora pachyrhizi infects many legume species, including soybeans (Glycine max) and the nonnative invasive species kudzu (Pueraria montana var. lobata). Our model predicts that epidemics are driven by the host species that is more abundant in the landscape. In managed landscapes, this will generally be the domesticated host. However, many pathogens overwinter on a wild host, which acts as the source of initial inoculum at the start of the growing season. Our model predicts that very low local densities of infected wild hosts, surviving in landscape patches separate from the domesticated host, are sufficient to initiate epidemics in the domesticated host, such that managing epidemics by reducing wild host local density may not be feasible. In contrast, managing to reduce pathogen infection of a domesticated host can reduce disease impacts on wild host populations.

Devosia yakushimensis sp. nov., isolated from root nodules of Pueraria lobata (Willd.) Ohwi.

A Gram-negative, strictly aerobic bacterium, comprising non-endospore-forming motile rods (1.2-2.0 microm x 0.4-0.6 microm) with polar flagellae was isolated from root nodules of the leguminous plant Pueraria lobata (Willd.) Ohwi growing on the coast of Yakushima Island, Kagoshima Prefecture, Japan. The novel strain, designated Yak96B(T), grew at an optimum pH of 7.0 and an optimum temperature of 28 degrees C. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the new strain was closely related to Devosia neptuniae J1(T) and Devosia chinhatensis IPL18(T), with sequence similarities of 98.1 % and 97.8 %, respectively. However, the DNA-DNA relatedness values of strain Yak96B(T) with D. neptuniae LMG 21357(T) and D. chinhatensis CCM 7426(T) were 53.6 % and 34 %, respectively. The DNA G+C content of strain Yak96B(T) was 65.3 mol%, the predominant isoprenoid quinone was Q10 (85 %) and the polar lipids were phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids (>5 %) were 11-methyl C(18 : 1)omega7c (35.0 %), C(16 : 0) (22.4 %), C(18 : 1)omega7c (21.8 %), C(19 : 0) cyclo omega8c (6.8 %) and C(18 : 0) (5.4 %). The infection/nodulation test was negative and nifH and nodD genes were not detected. Based on its chemotaxonomic and physiological characteristics, strain Yak96B(T) represents a novel species of the genus Devosia, for which the name Devosia yakushimensis sp. nov. is proposed. The type strain is Yak96B(T) (=KCTC 22147(T)=NBRC 103855(T)=LMG 24299(T)).

Isolation and characterization of nonrhizobial plant growth promoting bacteria from nodules of Kudzu (Pueraria thunbergiana) and their effect on wheat seedling growth.

The leguminous vine Kudzu (Pueraria thunbergiana) is an introduction into the N. W. Himalayan region of India. Despite its value as a fodder and cover crop, little is known about the nature of the nodule microflora. In an attempt to study the nodule bacteria, we isolated and characterized three nonrhizobial plant growth promoting bacteria from surface sterilized nodules of Kudzu. Based on the sequencing of the 16 S r RNA gene, the isolates were designated as Bacillus thuringiensis KR-1, Enterobacter asburiae KR-3, and Serratia marcescens KR-4. Crystalline bodies were detected in the isolate KR-1, confirming its identity as B. thuringiensis. Under in vitro conditions, all three isolates were found to produce indole acetic acid. Other plant growth promotion attributes such as P solubilization, hydrogen cyanide production, and ammonia production varied among the isolates. All of the three isolates promoted growth and positively influenced nutrient uptake parameters of wheat seedlings.

[Primary study on acid tolerance mechanism of a wild aciduric Rhizobium strain isolated from Pueraria lobata].

An aciduric Rhizobium strain, named as PR389, was isolated from the nodule of wild Pueraria lobata which grew in yellow soil (pH 4.6) on the Jin-yun Mountain in Bei-bei, Chong-qing city. The isolated strain, which could grow under pH 4.6 distinct from the optimal pH 6.5-7.5 for rhizobium, showed some typical features of aciduric rhizobium. This was also confirmed by the proton flux assay. Compared to the acid-sensitive Rhizobium strain PR21, the cell membrane of PR389 could hold back excessive H entering cell. This feature can protect PR389 from harm of acid. In the test of acid tolerance, the aciduric ability of strain PR389 under low acidic (pH 3.8) was restrained by antibiotic chloramphenicol. It was speculated that special proteins in the cells of PR389 could be induced and synthesized in acidic environment.

Genetic transformation of Pueraria phaseoloides with Agrobacterium rhizogenes and puerarin production in hairy roots.

An efficient transformation system for the medicinal plant Pueraria phaseoloides was established by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots could be obtained directly from the cut edges of petioles of leaf explants or via callus 10 days after inoculation with the bacteria. The highest frequency of explant transformation by A. rhizogenes ATCC15834 was about 70% after infection for 30 days. Hairy roots could grow rapidly on solid, growth regulator-free Murashige and Skoog medium and had characteristics of transformed roots such as fast growth and high lateral branching. Paper electrophoresis revealed that bacteria-free hairy roots of P. phaseoloides could synthesize agropine and mannopine. The polymerase chain reaction amplification of rooting locus genes showed that left-hand transferred DNA of the root inducing plasmid of A. rhizogenes was inserted into the genome of transformed P. phaseoloides hairy roots. The content of puerarin in hairy roots reached a level of 1.190 mg/g dry weight and was 1.067 times the content in the roots of untransformed plants.

[Culture of hairy roots in Pueraria phaseoloides and its puerarin production].

An efficient transformation system for genetic transformation of medicinal plant, Pueraria phaseoloides was developed, by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots could be obtained via callus from the cut edges of leaf explants of P. phaseoloides 20 days after inoculation with agrobacterium. 35 days after infection, the percentage of rooted leaf explants was about 85%. Hairy roots could have an autonomous growth on solid or liquid growth regulator-free MS medium but grew more rapidly and formed no callus during culture in liquid medium. The transformation of hairy roots was confirmed by PCR amplification of rolB and rolC genes of Ri plasmid from A. rhizogenes. To investigate the physiological difference between solid and liquid culture, the mitochondrial membrane potential in hairy roots cultured for 15 days in solid and liquid medium were also detected by the method of fluorescence labeling of 3,3'-dipropylthiadicarbocyanide iodide. The results showed mitochondrial membrane potential of hairy roots in liquid medium was 11.8 times higher than that on solid medium. The content of puerarin in hairy roots reached a level of 1.190 mg/g.dry.wt and was 2.5 times as much as that in the roots of P. phaseoloides seedlings and was also 1.067 times as much as that in the crude drug of several year-old Pueraria roots. Our experiments have laid a foundation for large-scale production of puerarin in P. phaseoloides hairy roots.

Macrocyclic trichothecenes are undetectable in kudzu (Pueraria montana) plants treated with a high-producing isolate of Myrothecium verrucaria.

Myrothecium verrucaria was found to be an effective pathogen against kudzu grown in the greenhouse and the field. M. verrucaria produced large amounts of macrocyclic trichothecenes when cultured on solid rice medium, including epiroridin E (16.8 mg/g crude extract), epiisororidin E (1 mg/g), roridin E (8.7 mg/g), roridin H (31.3 mg/g), trichoverrin A (0.6 mg/g), trichoverrin B (0.1 mg/g), verrucarin A (37.4 mg/g), and verrucarin J (2.2 mg/g). Most of these toxins were also isolated from M. verrucaria spores and mycelia grown on potato dextrose agar medium, including epiroridin E (32.3 mg/g), epiisororidin E (28.6 mg/g), roridin E (0 mg/g), roridin H (60 mg/g), trichoverrin A (1.3 mg/g), trichoverrin B (1.8 mg/g), verrucarin A (13.8 mg/g), and verrucarin J (131 mg/g). When M. verrucaria was cultured on liquid media, the numbers but not the amounts of toxins decreased. Only epiroridin E (28.3 mg/g), epiisororidin E (29.6 mg/g), verrucarin B (195 mg/g) and verrucarin J (52.6 mg/g) were measured when the fungus was cultured on cornsteep medium. On soyflour-cornmeal broth M. verrucaria produced several toxins, including epiroridin E (58.1 mg/g), epiisororidin E (5.8 mg/g), verrucarin B (29.9 mg/g) and verrucarin J (32 mg/g). In contrast, no macrocyclic trichothecenes were detected by HPLC analysis of plant tissues of kudzu, sicklepod, and soybean treated with aqueous suspensions of M. verrucaria spores formulated with a surfactant. Chloroform-methanol extracts of kudzu leaves and stems treated with M. verrucaria spores were less cytotoxic to four cultured mammalian cell lines than the corresponding extracts from control plants. Purified macrocyclic trichothecenes (verrucarin A and T-2 toxin) were very cytotoxic to the same cell lines (< or = 2 ng/ml). These results show that neither intact macrocyclic trichothecenes nor toxic metabolites could be detected in plant tissues after treatment with M. verrucaria spores. These results argue for both safety and efficacy for the use of M. verrucaria in biological control of kudzu and other noxious weeds, and support proceeding to animal feeding trials for further evaluation of safety.