Conidiation of Hirsutella thompsonii var. synnematosa in submerged culture

Fourteen monosporal isolates of Hirsutella thompsonii grew vegetatively in various liquid media producing typical phialidic-like conidiophores. Hirsutella thompsonii var. synnematosa from Ivory Coast (HtIC) was the only pathotype which produced true conidia in submerged culture. HtIC began producing conidia after 3 days incubation reaching a peak ranging from 6.8 × 10⁵ to 9.7 × 10⁷ conidia/ml between 6 and 11 days. A concentration of 10 g/liter of corn steep liquor and 0.2% Tween 80 were essential for maximum conidiation. Submerged conidia had a smooth but somewhat rugose conidial walls, whereas aerially formed conidia were distinctly verrucose. Germination of submerged conidia ranged from 5.2 to 12.9% and were virulent causing 32.5% infection to adult citrus rust mites when sprayed on citrus foliage at 1.2 × 10⁹ conidia/ml.     

Lack ofPer os toxicity or pathogenicity in rats fed the fungusHirsutella thompsonii

Symptoms of toxicity or pathogenicity were not observed in white rats fed either whole-culture broth or mycelia of the parasitic fungusHirsutella thompsonii. This fungus is presently under consideration as a microbial control agent of the citrus rust mite,Phyllocoptruta oleivora.     

Hirsutella thompsonii a fungal parasite of the citrus rust mitePhyllocoptruta oleivora in the Rio Grande Valley of Texas

Hirsutella thompsonii, a hyphomycetous fungus, was found attacking the citrus rust mitePhyllocoptruta oleivora for the first time in May, 1972, in the Lower Rio Grande Valley of Texas. Sharp reductions in mite populations occurred during this period. The fungus was isolated from mites collected from citrus fruits and leaves. The parasitic nature and the distribution of the pathogen are described.     

A new fungal pathogen of the scavenger mite,Tydeus gloveri

A new fungal pathogen, Hirsutella tydeicola, was found causing epizootics in populations of the scavenger mite, Tydeus gloveri, during the summer of 1979 and 1980 on citrus in Florida. The fungus is described in association with its host using light and scanning electron microscopy. H. tydeicola is compared with a closely related species, H. thompsonii, a coexisting pathogen of the citrus rust mite. All attempts to isolate the fungus on various agar media failed.     

Susceptibility of Brevipalpus phoenicis to entomopathogenic fungi

The pathogenicity of 52 isolates from several fungus species was studied for the false spider mite Brevipalpus phoenicis. In addition, the main stages during the course of infection by Hirsutella thompsonii, by far the most virulent pathogen, were studied by means of light and electron microscopy. Adult mites were confined to arenas prepared with citrus leaves in acrylic dishes containing agar–water. Conidial suspensions containing 10⁸ conidia/ml were applied, except for H. thompsonii, where a concentration of 10⁷ conidia/ml was used. The H. thompsonii isolates caused higher mortality, with indices higher than 90%. Observations under the scanning electron microscope (SEM) were performed at 0, 6, 12, 24, 48, 72, and 120 h after application of a H. thompsonii suspension containing 10⁷ conidia/ml. Twenty-four hours after inoculation, H. thompsonii conidia were observed attached to the mite’s integument. The conidia germinated and penetrated through the base of the setae on the hysterosoma. Colonization occurred after 48 h, as evidenced by mortality. Conidiogenesis occurred after 120 h, with the development of mycelium and conidiophores emerging from the posterior and anterior parts of the mite.     

Effect of Aeration on Growth and Aflatoxin Production by Aspergillus flavus in Submerged Culture

Aspergillus flavus ATCC 15517 produced up to 212 mg per liter of total aflatoxin in submerged culture in aerated (3,000, 6,000, 9,000, and 12,000 ml/min) and agitated medium in 14-liter fermentors with 10 liters of medium consisting of 2% yeast extract and 10% sucrose. Aflatoxin production increased with time. A maximum of 212 mg/liter was produced at 9,000 ml/min aeration, whereas the yield decreased substantially at the lower aeration rates. Two other strains of A. flavus synthesized aflatoxin in smaller quantities.     

Utilization of Molasses Sugar for Lactic Acid Production by Lactobacillus delbrueckii subsp. delbrueckii Mutant Uc-3 in Batch Fermentation

Efficient lactic acid production from cane sugar molasses by Lactobacillus delbrueckii mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid fermentation using molasses was not significantly affected by yeast extract concentrations. The final lactic acid concentration increased with increases of molasses sugar concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter was obtained at a molasses sugar concentration of 190 g/liter with a productivity of 4.15 g/liter/h. Such a high concentration of lactic acid with high productivity from molasses has not been reported previously, and hence mutant Uc-3 could be a potential candidate for economical production of lactic acid from molasses at a commercial scale.     

Conidioogenous cell differences among mutant and wild-type pathotypes of Hirsutella thompsonii var. thompsonii

Two mutants with different conidiogenesis were isolated from wild phatotypes of Hirsutella thompsonii var. thompsonii collected from the citrus rust mite in Florida. One mutant (WSI) produces more typical single-neck phialides and conidia than the wild type. The other mutant (SS) was developmentally altered in producing condiogenous cells earlier than the wild type as well as phialides with multiple neck capable of conidiogenesis. The mutant (SS) appeared more virulent than mutant (WSI) and the wild type when bioassayed using the citrus rust mite as a host.     

Genetic diversity in <Emphasis Type="Italic">Hirsutella thompsonii</Emphasis> isolates based on random amplified polymorphic DNA analysis

Hirsutella thompsonii Fisher is a host-specific fungal pathogen of the diverse group of arthropods known as the Acari. It particularly affects eriophyid mites, and the identification of its isolates is a difficult task due to its pleomorphic nature. Seven isolates of H. thompsonii var. thompsonii and H. thompsonii var. synnematosa collected from various agro-climatic regions of India were subjected to PCR analysis using random decamer primers to differentiate between them. Random amplified polymorphic DNA analysis revealed that all of the isolates from the citrus rust mite, Phyllocoptruta oleivora Ashmead (Acari: Eriophyidae), clustered together, with the exception of one, HtCRMB. The only isolate of H. thompsonii from Aceria guerreronis keifer (Acari: Eriophydae), HtPDBC, clustered separately.     

Enhanced production of triterpenoid in submerged cultures of Antrodia cinnamomea with the addition of citrus peel extract

In recent years, Antrodia cinnamomea has become a well-known medicinal mushroom in Taiwan. Triterpenoids are considered one of the most biologically active components found in A. cinnamomea. The aim of this research is to investigate the feasibility of enhancing triterpenoid production in shake flask cultures of A. cinnamomea by adding citrus peel extract. As a result of its containing essential oils, citrus peel extract is inhibitory to mycelial growth. In the experiments, the appropriate adding time is determined to be on day 7. Of the various citrus peel extracts tested, tangerine proves to be the most effective in enhancing polyphenol and triterpenoid production. With an addition of 2 % (v/v), the content and production of total polyphenols rises from 5.95 mg/g DW of the control and 56.73 mg/L to 23.52 mg/g DW and 224.39 mg/L, respectively, on day 28. The production of triterpenoids also increases from 99.93 to 1,028.02 mg/L, for more than a tenfold increase. An optimal level of tangerine peel additive is determined to be around 4 %. Furthermore, when compared with the mycelia of the control culture, the profiles of the HPLC analysis show that the mycelia cultured with the tangerine-peel addition contain more kinds of triterpenoids. This study demonstrates that the addition of citrus peel extract effectively enhances the production of bioactive metabolites in the submerged cultures of A. cinnamomea.     

Study on the innocuity ofHirsutella thompsonii. I. Infectivity in mice and guinea pigs

The present work concerns the innocuity ofHirsutella thompsonii Fisher isolated in Cuba from a citrus rust mite,Phyllocoptruta oleivora Ashm. This phytopathogenic arachnide is an important pest of different citrus fruit plantations. The infectivity tests were performed in 220 animals (mice and guinea pigs) inoculated by intraperitoneal and subcutaneous injections. Animals showed macroscopic alterations such as nodules in the liver capsule, spleen, subcutaneous tissue; superficial fibrinous adhesions on these organs and hepatosplenomegaly. No fungi were recovered from organs at any time and only disintegrated hyphal elements were observed. The histopathological study disclosed a non-specific tissue reaction to a foreign body. The results obtained suggest the non-infectivity and innocuity ofHirsutella thompsonii Fisher in mice and guinea pigs.      

Utilization of Citrus by-products

Proceeds from the production oi by-products may spell the difference between profit and loss in the citrus canning industry. The three primary by-products of citrus are dried citrus pulp, molasses, and citrus peel oil. The most important utilization of citrus wastes is as stock feed, which consumes large quantities of dried pulp and citrus molasses. Citrus seed oil, alcohol, pectin, bland syrup, and feed yeast have been produced to a lesser extent.     

Pilot testing ofKloeckera apiculata for the biological control of postharvest diseases of citrus

The efficacy of the yeastKloeckera apiculata, strain 34-9, in controlling postharvest decay of citrus fruit was evaluated in small-scale and pilot tests in commercial packinghouse. Kloeckera apiculata grew efficiently on different media and maintained its antagonistic activity against spore germination ofPenicillium italicum. In small-scale experiments with citrus fruits dipped in the yeast cell suspension, the development of decay in citrus was effectively inhibited. The yeast was compatible with a mixture of low concentration of a commonly chemical fungicide. In packinghouse tests, combining the yeast with 40 mg/kg Carbendazim (MBC) resulted a reduction in the incidence of decay to a level equal to that of the commercial treatment of 200 mg/kg MBC. The efficacy of the strain 34-9 could also be maintained under packinghouse conditions at a cell concentration of the yeast antagonist as low as 10⁶ cells/ml. No significant difference in the efficacy ofK. apiculata was found in either the drench or the spray application systems tested in citrus packinghouse. Scanning electron microscopy revealed attachment of the yeast cells to the pathogen hyphae. The high antagonistic activity of strain 34-9 against citrus blue mould may be related to its capability to compete withPenicillium italicum, for space and nutrients and /or involvement of directly antagonist of the yeast on the fungus.     

Reduction of Legionella spp. in Water and in Soil by a Citrus Plant Extract Vapor

Legionnaires'' disease is a severe form of pneumonia caused by Legionella spp., organisms often isolated from environmental sources, including soil and water. Legionella spp. are capable of replicating intracellularly within free-living protozoa, and once this has occurred, Legionella is particularly resistant to disinfectants. Citrus essential oil (EO) vapors are effective antimicrobials against a range of microorganisms, with reductions of 5 log cells ml⁻¹ on a variety of surfaces. The aim of this investigation was to assess the efficacy of a citrus EO vapor against Legionella spp. in water and in soil systems. Reductions of viable cells of Legionella pneumophila, Legionella longbeachae, Legionella bozemanii, and an intra-amoebal culture of Legionella pneumophila (water system only) were assessed in soil and in water after exposure to a citrus EO vapor at concentrations ranging from 3.75 mg/liter air to 15g/liter air. Antimicrobial efficacy via different delivery systems (passive and active sintering of the vapor) was determined in water, and gas chromatography-mass spectrometry (GC-MS) analysis of the antimicrobial components (linalool, citral, and β-pinene) was conducted. There was up to a 5-log cells ml⁻¹ reduction in Legionella spp. in soil after exposure to the citrus EO vapors (15 mg/liter air). The most susceptible strain in water was L. pneumophila, with a 4-log cells ml⁻¹ reduction after 24 h via sintering (15 g/liter air). Sintering the vapor through water increased the presence of the antimicrobial components, with a 61% increase of linalool. Therefore, the appropriate method of delivery of an antimicrobial citrus EO vapor may go some way in controlling Legionella spp. from environmental sources.     

Effect of neem seed extract on feeding,growth, survival,and reproduction of Diaprepes abbreviatus (Coleoptera: Curculionidae)

A commercially available neem seed extract, Neemix 4.5, containing 4.5% azadirachtin (AZA), was assessed for biological activity against the root weevil Diaprepes abbreviatus (L.), an important exotic insect pest of Florida citrus. Laboratory bioassays against neonatal and 3-wk-old larvae fed sliced carrot treated with Neemix produced dose-dependent larval mortality and reduced fresh weights among survivors of treatments. The weight response was greater than the mortality response for both larval age groups. Neonates treated with 45 mg/liter AZA weighed 60% less than those in the control after 4 wk. Three-week-old larvae treated with 45 mg/ liter AZA weighed 30% less than those in the control after 5 wk. When neonates were exposed to insect diet incorporated with Neemix, reductions in larval survival and weight were observed at concentrations as low as 4.8 mg/liter AZA after 6 wk. Larval growth was inhibited by >97% with 42.9 mg/liter AZA in the diet. A soil drench containing 30 mg/liter AZA reduced the survival and weight gain of neonates added to potted citrus and provided protection to the roots in a greenhouse experiment. A concentration of 90 mg/liter AZA was required to provide protection of citrus roots against 4-wk-old larvae. Reproductive effects were observed when adult weevils were fed foliage treated with Neemix. The numbers of larvae hatching per egg mass were reduced by 27% and 68% at 30 and 90 mg/liter AZA, respectively. These results suggest that Neemix should be further evaluated for use in integrated pest management (IPM) programs of citrus.     

The role of native vegetation on infection rates of Calacarus heveae (Acari: Eriophyidae) by Hirsutella thompsonii (Ascomycota: Ophiocordycipitaceae)

Hirsutella thompsonii (Fischer) (Ascomycota: Ophiocordycipitaceae), a fungal pathogen, often causes high mortality in populations of Calacarus heveae Feres (Acari: Eriophyidae), an important pest mite in rubber tree plantations (Hevea brasiliensis Muell. Arg., Euphorbiaceae). However, the ecological and climatic factors regulating this host-pathogen system are poorly known. We compared fungal infections in agroforestry and traditional rubber plantations to evaluate the role of native vegetation and climatic factors on infection rates of C. heveae by H. thompsonii. While the prevalence of H. thompsonii was higher in managed rubber tree plantations, the abundance of C. heveae was about three times higher in traditional plantations. Abundance of C. heveae, agroecosystem management type and microclimatic variables were responsible for driving the infection rates of H. thompsonii. Native vegetation was a source for H. thompsonii and also modified the crop’s microclimate, which contributed to its maintenance in the crop fields. Therefore, appropriate management practices may enhance the effects of entomopathogens on conservative biological control of pest mites in agroforestry systems.     

Effects of Waterborne Copper, Cyanide, Ammonia, and Nitrite on Stress Parameters and Changes in Susceptibility to Saprolegniosis in Rainbow Trout (Oncorhynchus mykiss)

The effects of toxic exposures on the susceptibility of rainbow trout (Oncorhynchus mykiss) to saprolegniosis were evaluated. Fish were exposed to sublethal concentrations of copper (0.25 mg/liter), cyanide (0.07 mg/liter), ammonia (0.5 mg/liter), and nitrite (0.24 mg/liter) for 24 h. After exposure, the fish were challenged by Saprolegnia parasitica (3.6 x 10(sup6) zoospores per liter) for 10 min. Cortisol and cholesterol were used to indicate stress response. Similar increases of cortisol were found for the four tested chemicals. All fish with cortisol levels higher than 370 ng/ml developed the disease, while only 24% of the fish with cortisol levels lower than 370 ng/ml were infected. Cholesterol levels remained unchanged after toxic exposure. Increased susceptibilities to the pathogen were observed for ammonia (71%), copper (57%), nitrite (50%), and cyanide (33%). The increases in susceptibility as a result of cyanide and nitrite exposure could be explained by the stress response. For copper and ammonia, the combination of two different effects, the stress response and specific impairments of the defense mechanism of trout against saprolegniosis, should be considered.     

Stimulation of Sanguinarine Production by Combined Fungal Elicitation and Hormonal Deprivation in Cell Suspension Cultures of Papaver bracteatum

Fungal elicitor preparations from either homogenized mycelia of Dendryphion penicillatum (Cda.) Fr., a specific pathogen of Papaver species, or conidia of Verticillium dahliae Kleb., a general pathogen, were added to 14-day-old suspension cultures of Papaver bracteatum. Plant tissue cultures were grown either in the presence or absence of 0.1 milligram of 2,4-dichlorophenoxyacetic acid per liter and 0.5 milligram of 6-benzylam-inopurine per liter. Dendryphion extracts elicited an accumulation of the benzophenanthridine alkaloid, sanguinarine, which was not greatly influenced by hormone deprivation. Millimolar concentrations of dopamine were detected under all conditions. Thebaine was found when cells were cultured in hormone-free media, but it was not elicitor dose dependent. Verticillium-elicited cultures accumulated sanguinarine in an elicitor-dose-dependent manner only under conditions of hormonal deprivation, resulting in an elevation of sanguinarine levels 5- to 500-fold greater than controls (2-10% dry weight). Most of the sanguinarine accumulated in the medium (23 milligrams per liter), with 85% of the alkaloid associated with a 100g sedimenting fraction that, upon light microscopic inspection, proved to be devoid of cells. In bioassays, sanguinarine showed significant biological activity at concentrations as low as 5 to 10 micrograms per milliliter against three general plant pathogens, Verticillium dahliae, Botrytis cinerea Pers. ex Fr., and Rhizoctonia solani Kuehn. Dendryphion was less affected by sanguinarine addition and displayed an ability to metabolize the alkaloid as evidenced by its loss from the media, subsequent accumulation in the mycelia, and ultimate disappearance over a 48-hour period. By comparison, dopamine and thebaine were less toxic to the general plant pathogens.     

Enhancement of β-Carotene Synthesis by Citrus Products

β-Ionone, a stimulatory compound in the microbiological production of β-carotene by mated cultures of Blakeslea trispora, could be replaced with low-cost agricultural by-products (citrus oils, citrus pulp, or citrus molasses) with as good or better carotene yields. Peak yields (81 to 129 mg of carotene per g of dry solids) were achieved in 5 days. The various citrus products tested did not change the pigments produced; all trans-β-carotene remained the pre-dominant pigment. The acid-hydrolyzed soybean meal and corn used in previous production media could be replaced with unhydrolyzed cottonseed embryo meal and corn in a medium that also contained a natural lipid, deodorized kerosene, nonionic detergent, and a precursor.     

Evaluation of phloxine B as a possible control agent against citrus thrips (Thysanoptera: Thripidae)

We evaluated the toxicity of phloxine B photoactive dye combined with a cane molasses bait against citrus thrips, Scirtothrips citri (Moulton). Laboratory bioassays conducted under artificial light showed that thrips mortality followed a log-dose probit-response model with an estimated LC50 of 0.0079% dye. Diluted cane molasses plus 0.01% phloxine B then was used as a standard for comparison of eight additional baits, including three formulations of concentrated citrus peel liquor (CCPL1-3). Citrus thrips mortality ranked highest to lowest with CCPL1 and CCPL3 > CCPL2, Mo-Bait and cane molasses > concentrated beet molasses, concentrated cane molasses, hemicellulose extract, and whey. Several commercial surfactants were tested to see if their addition to the standard increased efficacy. Hyper-Active, Kinetic, and Tween 60 at 1% and Cohort, Hyper-Active, Kinetic, and Silwet at 0.25% when added to the standard, reduced citrus thrips mortality, whereas Tween 60 and Dyne-Amic at 0.25% had no effect. Cane molasses with one or 5% phloxine B dye and CCPL1 with 1% dye were sprayed on citrus trees and allowed to weather in the field. Laboratory bioassays conducted after leaves had weathered for up to 8 d indicated that bait-dye toxicity was persistent. Possible use of the bait-dye mixture in commercial control of citrus thrips is discussed.