Shift of Bacterial Community in Synanthropic Mite Tyrophagus putrescentiae Induced by Fusarium Fungal Diet

Background

Tyrophagus putrescentiae (Acari: Astigmata) and Fusarium sp. co-occur in poorly managed grain. In a laboratory experiment, mite grazing resulted in significant reduction of fungal mycelium on cultivation plates. The destruction of mycelium appeared to be a result of an interaction between the mites, fungi and associated bacteria.

Methodology and Principal Findings

A laboratory experiment was performed to simulate a situation of grain multiinfested by mites and Fusarium fungi. Changes of mite-associated bacterial community in T. putrescentiae were described in 3 habitats: (i) T. putrescentiae mites from a rearing diet prior to their transfer to fungal diet; (ii) fungal mycelium before mite introduction; (iii) mites after 7 day diet of each Fusarium avenaceum, F. culmorum, F. poae and F. verticillioides. Bacterial communities were characterized by 16 S rRNA gene sequencing. In total, 157 nearly full-length 16 S rRNA gene sequences from 9 samples representing selected habitats were analyzed. In the mites, the shift from rearing to fungal diet caused changes in mite associated bacterial community. A diverse bacterial community was associated with mites feeding on F. avenaceum, while feeding on the other three Fusarium spp. led to selection of a community dominated by Bacillaceae.

Conclusions/Significance

The work demonstrated changes of bacterial community associated with T. putrescentiae after shift to fungal diets suggesting selection for Bacillaceae species known as chitinase producers, which might participate in the fungal mycelium hydrolysis.     

The effect of stored barley cultivars, temperature and humidity on population increase of Acarus siro, Lepidoglyphus destructor and Tyrophagus putrescentiae

The rate of population increase of three mite species, Acarus siro (L.), Lepidoglyphus destructor (Schrank) and Tyrophagus putrescentiae (Schrank), was studied on various types of barley and at various combinations of temperature and humidity. The mites were added into the chambers and incubated for 21 days on seven different kinds of barley coming from four sites, including six cultivars and a mixture. The population increase of all species was higher on the mixture than on any other cultivar, except for Sebastian and Calgary. The increase of mites was studied at constant temperatures ranging from 5 to 35 °C and relative humidity (RH) ranging from 50 to 90 %. Positive rate of increase was found above 70 % RH for all species. The optimal humidity was at 85 % RH for A. siro and L. destructor and at 90 % RH for T. putrescentiae. As concerns the temperature, positive rate of increase was found at temperatures higher than 10, 15 and 20 °C for A. siro, L. destructor and T. putrescentiae, respectively. The temperature optima were at 23, 25, and 30 °C for A. siro, L. destructor and T. putrescentiae, respectively. Model estimated on laboratory data was then fitted to temperature and humidity records from August to November in the Czech grain store. Estimated population rate of increase was rarely positive: for A. siro it was for 24 %, for L. destructor for only 1 % and for T. putrescentiae for only 7 % days of the study period. It is concluded that in the climatic conditions of the Czech Republic the population increase of three mite pests is negligible during autumn and winter.     

Real-time PCR detection and quantification of Fusarium poae,F. graminearum,F. sporotrichioides and F. langsethiae in cereal grains in Finland and Russia

Abstract

TaqMan real-time quantitative PCR assays were developed for the accurate detection and quantification of DNA from Fusarium poae and F. graminearum species, which are able to produce trichothecenes. These and other PCR assays were used for the quantification of trichothecene-producing Fusarium fungi in cereal grains. A correlation was found between the levels of F. poae DNA and nivalenol and enniatins in barley and between the levels of F. graminearum DNA and deoxynivalenol in oats. The correlations between F. poae DNA and nivalenol and F. graminearum DNA and deoxynivalenol levels were higher than those between these mycotoxins and morphologically determined F. poae and F. graminearum/F. culmorum contamination levels. The use of F. poae specific primers and probe together with F. sporotrichioides/F. langsethiae specific primers and probe in a multiplex qPCR assay yielded results in accordance with those obtained using these primers and probes separately.     

Temperature-dependent population growth of three species of stored product mites (Acari: Acaridida)

The pest potential of stored product mites depends on the reproduction rate that is affected by the environmental conditions. In this study we investigated the effect of temperature, ranging from 5 to 35°C, on the population growth of three important mite species, Acarus siro, Tyrophagus putrescentiae and Auleroglyphus ovatus at 85% r.h. Starting with 10 individuals the population increase of mites was observed after 3 weeks of cultivation, or after 6 weeks for those kept at low temperatures (5, 10, 12.5, and 15°C). The rate of increase was calculated for each temperature and species. The obtained data were fitted with polynomial models. The mite population growth rates increased with increasing moderate temperatures until 25°C, when r _m -values were 0.179, 0.177 and 0.190 for A. siro, A. ovatus and T. putrescentiae, respectively. The lower development threshold was 10.2°C in all three species. Estimated upper temperature threshold was higher in T. putrescentiae (49°C) than in A. siro and A. ovatus (38°C). Simulation of the rate of population increase under ideal conditions, using real temperature records obtained from Czech grain stores, showed that the pest mite populations increase only during 3.5 months within a typical 9-month storage season in Central Europe. These results indicate that control of mites, be it chemical, physical or biological, is recommended during the months when allergens and pests are produced, i.e. from September to mid November and in May.     

A study ofTyrophagus putrescentiae (Acari: Acaridae) as a facultative predator of southern corn rootworm eggs

An acarid mite,Tyrophagus putrescentiae (Schrank), was found to feed on southern corn rootworm (SCR) eggs in peanut and corn agroecosystems. In laboratory studies,T. putrescentiae eggs were generally oviposited directly on SCR eggs, and immatures fed only on previously damaged SCR eggs, depending upon adults to penetrate the chorion of SCR eggs. Individual adultT. putrescentiae significantly reduced (P<0.05) the number of larvae to eclose from 40 SCR eggs. Initial population levels of 1, 2, 5, 10, and 20 adult mites reduced the SCR egg population by 19, 43, 69, 92, and 98% respectively. Mites were attracted to SCR eggs from a distance of 8 cm, with significantly (P<0.001, ² test) more mites found in containers which included SCR eggs as compared with containers without SCR eggs. In greenhouse experiments, significantly (P<0.05) fewer SCR adults emerged from soil-filled containers which included 25T. putrescentiae and 100 SCR eggs, as compared with containers which had only SCR eggs. We suggest thatT.putrescentiae may be an important biological control agent of the SCR in certain agroecosystems.     

Occurrence of toxigenic strains of Fusarium in maize and barley in Germany

Summary A survey was made of maize and barley in Germany for the occurrence of toxigenic strains of Fusarium and of the mycotoxins produced in culture by these strains.The following 6 species of Fusarium were found: F. avenaceum, F. culmorum, F. equiseti, F.oxysporum, F. poae, and F. tricinctum. The species most commonly isolated from bird-damaged maize ears was F. avenaceum while F. culmorum was consistently isolated from maize stem rot. The predominant species in barley grain was F. poae while F. avenaceum, F. culmorum, and F. tricinctum were also isolated frequently.Cultures on autoclaved maize of all the Fusarium strains were assayed for toxicity by feeding to 1-day-old chickens for 14 days. Some strains of F. avenaceum, F. culmorum, F. equiseti, and F. oxysporum proved to be acutely toxic to chickens and caused mortality as well as marked reductions in weight gain and feed consumption. All the strains of F. poae and F. tricinctum had a low degree of toxicity.Culture material of all the strains were analyzed for the presence of 11 known Fusarium mycotoxins. The following 4 mycotoxins were detected in the strains examined: moniliformin in 9 out of 9 F. avenaceum strains (2 to 760 ppm) and in the single strain of F. oxysporum (1150 ppm); zearalenone in 4 out of 5 F. culmorum strains (320 to 1400 ppm); deoxynivalenol in 3 out of 5 F. culmorum strains.(1 to 15 ppm); and acetyldeoxynivalenol (1 to 2 ppm) in 3 out of 5 F. culmorum strains. This is the first report of moniliformin production by F. avenaceum and F. oxysporum and also the first report of the occurrence of moniliformin-producing Fusarium strains in Europe.     

Variation in 8-ketotrichothecenes and zearalenone production by Fusarium graminearum isolates from corn and barley in Korea

A total of 214 Fusarium graminearum isolates were obtained from corn and barley which were collected from Kangwon province and the southern part of Korea, respectively, and were tested for 8-ketotrichothecenes and zearalenone (ZEA) production on rice grains. The incidences of trichothecene production by 105 isolates of F. graminearum from corn were 59.0% for deoxynivalenol (DON), 37.1% for 15-acetyldeoxynivalenol(15-ADON), 13.3% for 3-acetyldeoxynivalenol (3-ADON), 7.6% for 3,15-diacetyldeoxynivalenol (3,15-DADON), 20.0% for nivalenol (NIV), 6.7% for 4-acetylnivalenol (4-ANIV), and 1.0% for 4,15-diacetylnivalenol (4,15-DANIV). DON chemotypes frequently produced 15-ADON as the major isomer rather than 3-ADON and 9 of the 61 DON chemotypes produced low levels of NIV. On the other hand, the incidences of trichothecene production of 109 isolates by F. graminearum from barley were 24.8% for DON, 72.5% for NIV, 62.4% for 4-ANIV, and 10.1% for 4,15-DANIV. Of these isolates, 78 were NIV chemotypes and only one isolate produced DON and 3-ADON as major toxins. In addition, 26 of the 78 NIV chemotypes produced low levels of DON. ZEA was frequently produced by the trichothecene-producing isolates and the incidences of ZEA were 51.4% and 31.2% for the isolates from corn and barley, respectively. There was a great regional difference in trichothecene production by F. graminearum isolates between corn- and barley-producing areas in Korea.     

Fusarium species and their mycotoxins in infected corn in Italy

Surveys of corn (infected plants and commercial kernels) forFusarium species and their mycotoxins were carried out on samples collected all over Italy and from some European and mediterranean countries.Investigations on samples of corn stalk and ear rot standing in the field, mainly collected in southern Italy, proved to be contaminated with zearalenone (ZON), zearalenols (ZOL), and deoxynivalenol (DON). TheFusarium species most frequently isolated, and their recorded toxigenic capability (in parentheses), were:F. moniliforme;F. culmorum (ZON, ZOL, DON, 3AcDON);F. equiseti (ZON, ZOL); andF. proliferatum (MF). Along with these species,F. graminearum group 2 (ZON, DON and/or 3AcDON or 15AcDON);F. chlamydosporum;F. acuminatum (type-A trichothecene derivatives); andF. semitectum were often found to be associated.F. heterosporum (ZON, ZOL);F. solani;F. crookwellense (ZON, ZOL, FUS, NIV);F. oxysporum (MF);F. avenaceum (MF);F. sporotrichioides (T-2 toxin and derivatives); andF. poae (DAS, MAS) were occasionally isolated.     

Use of vapor pressure deficit to predict humidity and temperature effects on the mortality of mold mites, <Emphasis Type="Italic">Tyrophagus putrescentiae</Emphasis>

The mold mite, Tyrophagus putrescentiae (Shrank), frequently infests a variety of stored food products in ideal, but rather limited conditions. Major factors limiting survival of this mite are the temperature and humidity imposed on T. putrescentiae as it develops within and disperses among sites. However, since relative humidity is dependent upon air temperature, determining survivability in a habitat can be difficult in the presence of structural temperature variations. Vapor pressure deficit (VPD) provides a method of combining both relative humidity and temperature into a single number that can be used to determine conditions detrimental to mite survival. This study utilized a bioassay format to measure mortality of T. putrescentiae when exposed to a range of seven temperatures (5–35°C), 10 relative humidities (0–100% RH), 17 exposure times (0.5–240 h), with and without food. With these combinations of temperature and RH, mortality curves (mortality versus time) that displayed a sigmoidal relationship were used to calculate LT₅₀ and LT₉₀ estimates. These mortality estimates were then regressed on their associated VPD and the resulting regressions (LT₅₀ and LT₉₀) were significant at P < 0.0001, and provided acceptable R ² values ≥0.83, regardless of whether food was present or not. At room temperature, threshold of VPD for T. putrescentiae development was below 8.2 mbar, this estimate being initially calculated from published values. For mites exposed to drier conditions, above 8.2 mbar, survival time was curtailed dependant on the magnitude of VPD. As the VPD exceeded 12 mbar, mites experienced substantial (>90%) mortality within 58 (33, 101) h; and further increasing VPD decreased the time of exposure to achieve mortality. This study demonstrates that making subtle changes in humidity or temperature to reach a target VPD may provide control of mite outbreaks and reduce areas inhabitable for T. putrescentiae. With the recent revision of the genus Tyrophagus (Fan and Zhang 2007), T. putrescentiae was split and the commonly encountered peridomestic mite was renamed T. communis. Voucher specimens of the species we used were identified as T. communis (B. OConnor, pers. comm.). However, there are current discussions as to which species name will be applied to the more common mite species (P. Klimov, pers. comm.). For the purposes of this paper we will continue to use T. putrescentiae.     

Pollen,mould mites and fungi: improvements to mass rearing ofTyphlodromus doreenae andAmblyseius victoriensis

Populations ofTyphlodromus doreenae Schicha on 18×14 cm arenas using pollen ofTypha orientalis Presl. as food, increased from an estimated 50–150 to 1,000–1,600 motiles per arena in the presence of mould mites,Tyrophagus putrescentiae (Schrank) and the fungusRhizopus stolonifer (Ehrenberg ex. Fr.) Lind.T. putrescentiae in association withR. stolonifer, developed pigmentation and were preyed on byT. doreenae which also became pigmented. UnpigmentedT. putrescentiae were not attacked byT. doreenae which remained pale coloured.T. doreenae died when supplied with the fungus alone. Bean plants dusted with pollen ofT. orientalis from a talcum powder dispenser supported a large population (7–12 stages/leaf) ofAmblyseius victoriensis (Womersley). These improvements to mass rearing ofT. doreenae andA. victoriensis are discussed with respect to their potential for commercialisation.     

Mites as Selective Fungal Carriers in Stored Grain Habitats

Mites are well documented as vectors of micromycetes in stored products. Since their vectoring capacity is low due to their small size, they can be serious vectors only where there is selective transfer of a high load of specific fungal species. Therefore the aim of our work was to find out whether the transfer of fungi is selective. Four kinds of stored seeds (wheat, poppy, lettuce, mustard) infested by storage mites were subjected to mycological analysis. We compared the spectrum of micromycete species isolated from different species of mites (Acarus siro, Lepidoglyphus destructor, Tyrophagus putrescentiae, Caloglyphus rhizoglyphoides and Cheyletus malaccensis) and various kinds of stored seeds. Fungi were separately isolated from (a) the surface of mites, (b) the mites' digestive tract (= faeces), and (c) stored seeds and were then cultivated and determined. The fungal transport via mites is selective. This conclusion is supported by (i) lower numbers of isolated fungal species from mites than from seeds; (ii) lower Shannon–Weaver diversity index in the fungal communities isolated from mites than from seeds; (iii) significant effect of mites/seeds as environmental variables on fungal presence in a redundancy analysis (RDA); (iv) differences in composition of isolated fungi between mite species shown by RDA. The results of our work support the hypothesis that mite–fungal interactions are dependent on mite species. The fungi attractive to mites seem to be dispersed more than others. The selectivity of fungal transport via mites enhances their pest importance.     

Impact of proteins and saccharides on mass production of Tyrophagus putrescentiae (Acari: Acaridae) and its predator Neoseiulus barkeri (Acari: Phytoseiidae)

Proteins and saccharides are the two most important nutrients of artificial insect diets. In this study, additional protein or saccharide sources were added to the diet, and their impact on the population increase of both the prey Tyrophagus putrescentiae Schrank (Acari: Acaridae) and the predator Neoseiulus barkeri Hughes (Acari: Phytoseiidae) was investigated. T. putrescentiae population increased by 319, 317 and 180 times within six weeks, when yeast powder, glucose or sugar was added to the basic wheat bran diet (diet mass: additive mass 10:3), respectively. However, T. putrescentiae population increased by only 70 times when reared on the basic diet. All three types of nutrients resulted in increased soluble saccharide level of mixed stages T. putrescentiae. Significant increase of soluble protein level was observed when yeast powder was added. When fed on T. putrescentiae reared on yeast powder, glucose or sugar added diets, the developmental duration of N. barkeri was shortened by 23, 23 and 33%, and the daily fecundity increased by 40, 20 and 27%, respectively. The proportion of N. barkeri female offspring was 64% when fed with T. putrescentiae reared on wheat bran, increased to 70% when yeast powder was added, and decreased to 59% and 58% when glucose and sugar was added, respectively. The commercial packaging requirement of N. barkeri is 80 mites per g. It generally takes 40 days from N. barkeri inoculation to reach this requirement, but this period was dramatically shortened to 20, 25 and 24 days when yeast powder, sugar and glucose were added to the diet of T. putrescentiae, respectively.     

The occurrence of culmorin and hydroxy-culmorins in cereals

Forty-five samples from 1988–1995 of naturally contaminated grain, barley, wheat and oats, three samples of mixed feed, and 16 samples of grain artificially inoculated with Fusarium culmorum during the flowering stage were analysed for deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-acetyl-DON), culmorin and hydroxy-culmorins. These compounds are secondary metabolites produced by the fungal species F. culmorum and F. graminearum. Acetonitrile-water extract of the samples was purified on a Mycosep^TM#225 column, derivetized using pentafluoropropionic anhydride (PFPA) and analysed by gas chromatography-mass spectrometry (GC-MS). The amount of each of culmorin, 5-, 12-, 14 and 15-hydroxy-culmorin and one unknown hydroxy-culmorin were determined relative to the amount of DON plus 3-acetyl DON for each sample. The ratio between the total amount of culmorin compounds and the DON compounds ranged from 0.14 to 1.07 in the samples. This study shows that there is a strong correlation between the amount of DON present in the grain and the amount of culmorin and hydroxy-culmorins present. The ratio of each of the culmorin compounds relative to the amount of DON compounds were in the same range in the grain artificially inoculated by F. culmorum as found in an earlier study for F. culmorum strains cultivated on rice, while the hydroxy-culmorin profile in the naturally contaminated grain was more similar to what was found for the F. graminearum cultures in the same study [1]. These results indicate that F. graminearum may be a relatively important source for DON in grain also in relatively cold areas. This revised version was published online in June 2006 with corrections to the Cover Date.     

SRAP as an Informative Molecular Marker to Study the Fusarium poae Genetic Variability

Fusarium poae is one of the Fusarium species isolated from grains associated with Fusarium head blight (FHB), whose occurrence has increased in the last years. In this study, a total of 105 F. poae isolates from Argentina, Belgium, Canada, England, Finland, France, Germany, Hungary, Italy, Luxembourg, Poland, Switzerland and Uruguay were evaluated using sequence‐related amplified polymorphism (SRAP) to analyse the capacity of this molecular marker to evaluate the F. poae genetic variability. The molecular analysis showed high intraspecific variability within F. poae isolates, and a partial relationship was revealed between variability and the host/geographic origin. Analysis of molecular variance (amova ) indicated a high genetic variability in the F. poae collection, with most of the genetic variability resulting from differences within, rather than between American and European populations. The analysis of sequenced SRAP fragments targets into hypothetical proteins from different Fusarium species showing that the SRAP technique not only allows studying F. poae genetic variability, but also targets coding regions into the F. poae genome. To our knowledge, this is the first report on genetic variability of F. poae using SRAP technique and also demonstrates the efficacy of this molecular marker to amplify open reading frames in fungus.     

Response to Fusarium culmorum inoculation in barley

In field tests replicated in 2004 and 2005, 32 cultivars of spring barley were assessed for resistance to Fusarium head blight (FHB) by single floret inoculation and spray inoculation with Fusarium culmorum (W. G. Smith) Sacc. It was found that the weather conditions in individual years affect to a large extent the progression of FHB and production of mycotoxin deoxynivalenol (DON). At the same time, in both years the cultivars reacted to F. culmorum infection similarly with respect to areas under disease progress curve (AUDPC) values and content of mycotoxin DON. Spraying inoculation led to stronger infection. The biggest differences in AUDPC values were observed between the cultivars Brise and Celinka, and weak reaction was found in the cultivars Kompakt and Madonna. The cultivars Kompakt and Tolar were most resistant towards FHB. In both monitored years the variety Ludan contained the lowest amounts of mycotoxin DON. Cultivars with high infection and low DON content (r = 0.78) showed weak positive relationship between resistance to FBH and accumulation of DON (concentration 70–200 mg/kg). This is the first information on FHB and in vivo concentrations of DON in certificated barley cultivars in Slovakia.     

Occurrence and distribution of <Emphasis Type="Italic">Microdochium nivale</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from barley,durum and soft wheat grains in France from 2000 to 2002

Fusarium Head Blight of small grain cereal is a disease of growing concern in Europe. Along with Microdochium nivale, several species of Fusarium may be associated with the disease, including species that are potentially toxigenic. This paper describes the results of a large scale survey of the variety and frequency of different Fusarium species and M. nivale in France. A total of 749 soft wheat, durum wheat and barley samples were collected and analyzed from 2000 to 2002. The most frequent species isolated were F.graminearum, F. avenaceum and F. poae. The frequency of F. poae seems to have increased while M.nivale and F. culmorum appear less frequent than previously described in France. Other Fusarium species detected in decreasing prevalence were F. tricinctum, F. equiseti, F. acuminatum, F. sambucinum, F.sporotrichioides, F. moniliforme, F. heterosporum, F. subglutinans and F. oxysporum. All the most frequent pathogenic species and also the less pathogenic ones were frequently associated with individual fields. The implications of these associations for the protection of cereals crops and for contamination by mycotoxins are discussed.     

Quantification of Fusarium culmorum in Wheat and Barley Tissues Using Real-Time PCR in Comparison with DON Content

A real‐time polymerase chain reaction (PCR) assay was developed for the specific detection of Fusarium culmorum in infected seeds. Primers and TaqMan minor groove binder probe were derived from the sequences of a F. culmorum specific PCR product. The specificity of the assay was confirmed by test in seven Fusarium species and 21 non‐Fusarium fungal species. With serial dilutions of purified genomic DNA from F. culmorum isolate B as the template, the detection limit of the assay was found to be 0.9 pg of fungal genomic DNA per reaction. A significant correlation ( = 0.982) and collinearity was found between DNA concentration and Ct (cycle threshold) values of real‐time PCR assay with serial diluted DNAs extracted from three seed samples with different deoxynivalenol (DON) content. Eight barley and nine wheat varieties infected by F. culmorum isolate B were evaluated in 1 (barley samples) and in 4 years (wheat samples). The results of real‐time PCR analysis and enzyme‐linked immunosorbent assay testing for DON content were compared and a significant correlation was found for barley samples (r² = 0.935). Concerning wheat we found rather complicated relationship between Ct values and DON contents influenced by environmental conditions of field trials. The real‐time PCR assay was found to be highly specific and sensitive. It could be used in phytopathological studies and praxis.     

Combination of the antifeedant bean flour and the predator Cheyletus malaccensis suppresses storage mites under laboratory conditions

Stored product mites are pests of serious economic and medical importance. Recently, it has been shown that the biological control of these pests based on the use of natural compounds and predators had a high potential for success. In this study, we investigated the suppression of three pest mites by combination of the predator Cheyletus malaccensis Oudemans, 1913 and bean flour (Phaseolus vulgaris), which is an antifeedant. Wheat grain (100 g) was contaminated with 100 individuals (Acarus siro (Linnaeus, 1758), Aleuroglyphus ovatus (Troupeau, 1878) or Tyrophagus putrescentiae (Schrank, 1781)). We added bean flour in concentrations of 0, 1, 5 and 10 g kg⁻¹ (wt/wt) and/or the predator with initial predator/prey ratios 0, 1/50, 1/25 and 1/10. The experiment was carried out under abiotic conditions that are optimal for pest development. After 21 days, pest and predators numbers were recorded. Application of flour significantly reduced populations of A. siro, slightly reduced populations of T. putrescentiae, the efficiency increased with increasing concentration. A. ovatus was not affect by bean flour. Populations of all mite species were successfully reduced by the sole addition of C. malaccensis, with a higher efficiency at higher Cheyletus ratios. The additive effect of the predator and flour was mainly apparent in the reduction of T. putrescientae. The population of C. malaccensis was not affected by the presence of bean flour. It is therefore recommended to use bean flour for the reduction of A. siro, a combination of bean flour and Cheyletus for the reduction of T. putrescientae, and only Cheyletus for the reduction of A. ovatus. Handling Editor: Arne Janssen.