Dorothy M. Hinton

Enterobacter cloacae is an endophytic symbiont of corn

The bacteriumEnterobacter cloacae is presently used for biocontrol of postharvest diseases of fruits and vegetables and as a preplant seed treatment for suppression of damping-off. This bacterium has apparent affinities for several grass species, but it is not considered to be an endophyte. While screening corn for fungi and bacteria with potential for biocontrol of various corn diseases, the surface-sterilized kernels of one unknown Italian corn cultivar produced fungus-free corn seedlings with roots endophytically infected byE. cloacae. This paper describes the microscopic nature ofE. cloacae RRC 101 with corn, and the in vitro control ofFusarium moniliforme and other fungi with this bacterium. Light and electron microscopy determined that this isolate ofE. cloacae was biologically associated with corn seedling roots, where it was distributed intercellularly within the cortex and stele. This is a first report of a strain of this bacterium as an endophytic symbiont of roots. Following a topical application ofE. cloacae to kernels, and upon germination this bacterium readily infected roots of two other corn cultivars. The bacterium was observed within the endosperm of germinating corn seedling, but germination was not affected. Further, the bacterium was isolated from leaves and stems of 3- to 6-week-old seedlings indicating that the above ground portions of corn were also colonized. There was no evidence of damage to cells of the root during a three to four week observation period. This bacterium was antagonistic to several isolates of the corn pathogenFusarium moniliforme, and to two other species of fungi, all of which produce mycotoxins on corn.

Bacterial endophytes: The endophytic niche, its occupants, and its utility

Endophytic species of bacteria offer an enormous potential for protection and increased agronomic performance of plants. The diversity of bacterial endophytes guarantees that there are endophytes capable of forming compatible association with all agronomically important plants, including monocots and dicots. Fitness of endophytically infected plants is increased both at inter- and intraspecific levels of competition. These bacterial offer the advantages of being sequestered within plants and as long as the plant has a favorable environment to grow, the bacteria contained within it will continue to grow affording the plant continued protection throughout its growth period. The bacteria are endosymbionts, and behave in most instances the relationships are mutualists. The habit is the intercellular spaces of plants, a large area of interconnected spaces that contain high levels of carbohydrates, amino acids, and inorganic nutrients, which serve the purpose of supporting the growth of intercellular bacteria. The intercellular niche is a novel compartment from which the plant can be protected, offering numerous advantages over conventional pesticides. In addition to biocontrol uses, endophytic bacteria offer potential for surrogate transformation of plants resulting in increased nutritional qualities or novel pesticides, as well as the recently emerging use for phytoremediation of soil and water pollutants. Endophytic bacteria are defined as bacteria that colonize healthy plant tissue without causing obvious symptoms in or produce obvious injury to the host. Bacterial endophytes colonize a large number of plants, but because they are symptomless they cannot be detected, and can only be detected by isolating such bacteria from surface sterilized plants placed onto bacterial agar. This procedure foretells the difficulty in providing a valid estimate of the numbers of bacterial endophytes and their roles in planta. Bacterial endophytes actively colonize plant tissues, establish long-term associations, actually lifelong natural associations, and are not in generally organ-specific. Thus, they may be isolated from roots, leaves, and stem, and a few from inflorescences and fruits. Excluded from the group of bacterial endophytes are those that are associated with plants as latent infections and or colonize senescent plant tissue as evidenced by the production of macroscopic signs of diseases. Therefore, a bacterial endophyte is a microbe that lives in association with plants forming a symptomless intercellular biotrophic association (Fig. 1). Endophytic bacteria are distinguished from transient visitors that form plant associations as happenstances, which do not survive long. Bacterial endophytes as used here encompass the broader use of the narrowly defined term of Kado (1992) to include that of Quispel (1992), which defines bacterial endophytes to include

Isolation and Characterization of Leu7-Surfactin from the Endophytic Bacterium Bacillus mojavensis RRC 101, a Biocontrol Agent for Fusarium verticillioides

Bacillus mojavensis is an endophytic bacterium patented for control of fungal diseases in maize and other plants. Culture extracts and filtrates from this bacterium were antagonistic to the pathogenic and mycotoxic fungus Fusarium verticillioides. However, the identity of the inhibitory substance from extracts of this bacterium has not been determined. An HPLC-MS analysis of the culture filtrate showed a major ion peak that was identified as a cyclic lipopeptide. Furthermore, collisional ion dissociation (CID) analysis indicated that this lipopeptide was surfactin, a cyclic heptapeptide linked to a β-hydroxy fatty acid. A CID analysis of the peptide moiety was established by deduction and indicated that the peptide sequence consisted of two acidic amino acids and five hydrophobic amino acids with a sequence of Leu-Leu-Asp-Val-Leu-Leu-Glu. These spectra indicated that this bacterium produced Leu(7)-surfactin, which was toxic to F. verticillioides. Production of this cyclic lipopeptide is a characteristic of several species of Bacillus, but this is the first report of this very powerful biosurfactant from this endophytic species.

The black Aspergillus species of maize and peanuts and their potential for mycotoxin production.

The black spored fungi of the subgenera Circumdata, the section Nigri (=Aspergillus niger group) is reviewed relative to their production of mycotoxins and their effects on plants as pathogens. Molecular methods have revealed more than 18 cryptic species, of which several have been characterized as potential mycotoxin producers. Others are defined as benign relative to their ability to produce mycotoxins. However, these characterizations are based on in vitro culture and toxins production. Several can produce the ochratoxins that are toxic to livestock, poultry, and humans. The black aspergilli produce rots of grapes, maize, and numerous other fruits and grain and they are generally viewed as post-harvest pathogens. Data are review to suggest that black aspergilli, as so many others, are symptomless endophytes. These fungi and their mycotoxins contaminate several major grains, foodstuffs, and products made from them such as wine, and coffee. Evidence is presented that the black aspergilli are producers of other classes of mycotoxins such as the fumonisins, which are known carcinogenic and known prior investigations as being produced by the Fusarium species. Three species are identified in U.S. maize and peanuts as symptomless endophytes, which suggests the potential for concern as pathogens and as food safety hazards.

Identification and characterization of bacterial endophytes of rice

We isolated seven different bacteria from rice seedlings grown from surface sterilized seeds. Three were associated with the rice seed husk and the other four were growing endophytically within the seed. Microscopic studies revealed that the endophytes were concentrated in the root stele region. Some of the bacteria exhibited strong anti-fungal activity against Rhizoctonia solani, Pythium myriotylum, Guamannomyces graminis and Heterobasidium annosum.

Potential for control of seedling blight of wheat caused byFusarium graminearumand related species using the bacterial endophyteBacillus mojavensis

Abstract Fusarium-infected wheat seed decreases germination, seedling emergence, and causes post emergence seedling death, and can contribute to wheat scab and ear rot of maize, with consequent production of mycotoxins such as deoxynivalenol and zearalenone. Current seed treatments have proved ineffective in controlling seedling blight and scab. A patented endophytic bacterial strain, Bacillus mojavensis RRC 101, and several other strains of this species were studied to determine in vitro antagonism to some Fusarium species and to assess the potential of this bacterium to serve as an endophytic biocontrol for seedling blight of wheat produced by species within the F. graminearum complex, as well as other species of Fusarium. Seedling emergence and seed germination were two tests used as indicators of seedling blight. These tests were conducted in growth rooms with two wheat cultivars highly susceptible to scab, Norm and Pioneer 2552, and other cultivars with varying resistance to scab. The results indicated that all strains of this bacterium were antagonistic in vitro to the strains of F. graminearum and its seven related species, as well as four strains of F. pseudograminearum and the two strains of F. verticillioides. Germination of the highly scab susceptible cultivar 2552 was increased from 77 to 97% when planted in soil containing a mixed inoculum of F. graminearum and related species. Seedling emergence in the very susceptible wheat cultivar Norm increased from 20 to 82% when treated with the bacterium. The data indicated that inoculating wheat kernels with B. mojavensis reduced seedling blight of wheat produced by F. graminearum and related Fusarium species indicating the potential for this bacterium as a biocontrol under field condition.

Field performance of maize grown from Fusarium verticillioides-inoculated seed.

Fusarium verticillioides is an important fungus occupying dual roles in the maize plant. The fungus functions as an endophyte, a fungal/host interaction beneficial to the growth of some plants. At other times, the fungus may function as a mycotoxin producing pathogen. The advantages and/or disadvantages of the endophytic relationship must be established in order to target appropriate sites for controlling diseases and mycotoxins in maize. One possibility could be to ensure seed maize is fungal free prior to planting. Reciprocal inoculations were made with two fungal isolates on seed of two maize genotypes. Yield was measured at harvest by ear and seed characters and vegetative growth at one-month intervals for plant survival, height, weight and stem diameter. Yield and vegetative growth differed among mature plants only once based on seed inoculation status. In 1998, plant weight was reduced and seed weight per ear was increased for the dent maize, GT-MAS: gk, grown from F. verticillioides RRC 374- inoculated seed compared to other seed treatments. Most vegetative characters were reduced at the first collection for Silver Queen plants grown from F. verticillioides-inoculated seed in 1997 and 1999, but not in 1998. However, no significant differences occurred among mature Silver Queen plants during any of the three growing seasons. In conclusion, yield and vegetative growth of mature maize plants grown from F. verticillioides-inoculated seed were equal to or greater than plants grown from non-inoculated seed under south Georgia field conditions during 1997, 1998, and 1999.

Interactions of Bacillus mojavensis and Fusarium verticillioides with a Benzoxazolinone (BOA) and its Transformation Product, APO

The benzoxazolinones, specifically benzoxazolin-2(3H)-one (BOA), are important transformation products of the benzoxazinones that can serve as allelochemicals providing resistance to maize from pathogenic bacteria, fungi, and insects. However, maize pathogens such as Fusarium verticillioides are capable of detoxifying the benzoxazolinones to 2-aminophenol (AP), which is converted to the less toxic N-(2-hydroxyphenyl) malonamic acid (HPMA) and 2-acetamidophenol (HPAA). As biocontrol strategies that utilize a species of endophytic bacterium, Bacillus mojavensis, are considered efficacious as a control of this Fusarium species, the in vitro transformation and effects of BOA on growth of this bacterium was examined relative to its interaction with strains of F. verticillioides. The results showed that a red pigment was produced and accumulated only on BOA-amended media when wild type and the progeny of genetic crosses of F. verticillioides are cultured in the presence of the bacterium. The pigment was identified as 2-amino-3H-phenoxazin-3-one (APO), which is a stable product. The results indicate that the bacterium interacts with the fungus preventing the usual transformation of AP to the nontoxic HPMA, resulting in the accumulation of higher amounts of APO than when the fungus is cultured alone. APO is highly toxic to F. verticillioides and other organisms. Thus, an enhanced biocontrol is suggested by this in vitro study.

In planta reduction of maize seedling stalk lesions by the bacterial endophyte Bacillus mojavensis

Maize (Zea mays L.) is susceptible to infection by Fusarium verticillioides through autoinfection and alloinfection, resulting in diseases and contamination of maize kernels with the fumonisin mycotoxins. Attempts at controlling this fungus are currently being done with biocontrol agents such as bacteria, and this includes bacterial endophytes, such as Bacillus mojavensis . In addition to producing fumonisins, which are phytotoxic and mycotoxic, F. verticillioides also produces fusaric acid, which acts both as a phytotoxin and as an antibiotic. The question now is Can B. mojavensis reduce lesion development in maize during the alloinfection process, simulated by internode injection of the fungus? Mutant strains of B. mojavensis that tolerate fusaric acid were used in a growth room study to determine the development of stalk lesions, indicative of maize seedling blight, by co-inoculations with a wild-type strain of F. verticillioides and with non-fusaric acid producing mutants of F. verticillioides. Lesions were measured on 14-day-old maize stalks consisting of treatment groups inoculated with and without mutants and wild-type strains of bacteria and fungi. The results indicate that the fusaric-acid-tolerant B. mojavensis mutant reduced stalk lesions, suggesting an in planta role for this substance as an antibiotic. Further, lesion development occurred in maize infected with F. verticillioides mutants that do not produce fusaric acid, indicating a role for other phytotoxins, such as the fumonisins. Thus, additional pathological components should be examined before strains of B. mojavensis can be identified as being effective as a biocontrol agent, particularly for the control of seedling disease of maize.

Bacillus mojavensis: Its Endophytic Nature, the Surfactins, and Their Role in the Plant Response to Infection by Fusarium verticillioides

Bacterial endophytes are fundamentally important as natural components of most plants, wild and cultivated with strong ecological merits. These ancient associations are recently at the forefront of biological control strategies designed to circumvent the problems associated with pesticide uses, particularly on specific food crops. Bacterial endophytes form compatible associations that persist during the growing seasons, where several enhanced benefits are associated, suggesting that such associations are mutualistic. Several genera of bacteria are known as root associations, and only recently are species being identified as plant endophytes, occupying the entire plant axis in most cases. Bacillus mojavensis was discovered in maize kernels and later determined to be an endophyte with biocontrol potential due to its inhibition of the maize mycotoxic and pathogenic fungus Fusarium verticillioides, itself an endophyte. It was subsequently shown that this strain and others were inhibitory to most fungi, especially plant pathogenic species. Further, maize plants infected with B. mojavensis showed a marked improvement in foliage and root growth and development, disease protection, and mycotoxin reduction. Components of B. mojavensis-infected maize are reviewed relative to biocontrol of F. verticillioides and other endophytic fungi. The patented and other strains of this bacterium were recently reported as producers of the lipopeptide biosurfactant Leu7-surfactin. The chemistry, fermentation of the surfactins, and their uses, along with the essential features of surfactins required for fungal inhibition, are discussed. We also review the host–parasite relations of this bacterial endophyte, and its biochemical utility in an effort to bring attention to the potential qualities of B. mojavensis, and other bacterial endophytes for enhancers of plant growth and protectors of diseases.