Effect of inoculation method and plant growth medium on endophytic colonization of sorghum by the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

A study was conducted to determine the effect of inoculation method and plant growth medium on colonization of sorghum by an endophytic Beauveria bassiana. Colonization of leaves, stems, and roots by B. bassiana was assessed 20-days after application of the fungus. Although B. bassiana established as an endophyte in sorghum leaves, stems, and roots regardless of inoculation method (leaf, seed, or soil inoculation), plant growth medium (sterile soil, non-sterile soil, or vermiculite) apparently influenced colonization rates. Seed inoculation with conidia caused no stem or leaf colonization by the fungus in non-sterile soil but did result in substantial endophytic colonization in vermiculite and sterile soil. Leaf inoculation did not result in root colonization, regardless of plant growth medium. Endophytic colonization was greater in leaves and stems than roots. Endophytic colonization by B. bassiana had no adverse effects on the growth of sorghum plants. Leaf inoculation with a conidial suspension proved to be the best method to introduce B. bassiana into sorghum leaves for plants growing in either sterile or non-sterile soil. Further research should focus on the virulence of endophytic B. bassiana against sorghum stem borers.     

二种交配型球孢白僵菌对亚洲玉米螟的生态控制作用

利用生物种间互做关系抑制农业害虫的暴发是生物防治的重要手段。为探讨二种交配型内共生球孢白僵菌与玉米之间的互惠关系及其形成的共生体在亚洲玉米螟控制中的生态效应,以玉米为宿主植物,以球孢白僵菌孢子悬浮液进行灌根,在温室内构建了二种交配型(MAT1-1-1型,B5;MAT1-2-1型,B2)球孢白僵菌-玉米共生体,并研究了共生体对玉米的生长、对亚洲玉米螟的产卵选择和幼虫发育及其对球孢白僵菌生物学特性的影响。结果显示:通过叶片离体培养、ITS基因和交配型基因MAT检测,均能检测到白僵菌的内生定殖;MAT1-2-1型B2菌株定殖检出率高,MAT1-1-1型B5菌株在混合型接种中定殖有优势。回收后的球孢白僵菌菌落直径和毒力无显著性变化,但其产孢量都显著提高其中回收B5处理组来源菌株的产孢量提高最显著。接种过球孢白僵菌的玉米植株地上部生长速度、生物量和地下根系生物量均优于对照组,其中根系干重明显增加,而地上植株干重也相对增加。MAT1-1-1型菌株B5对共生体玉米植株地上高度促生长贡献明显;MAT1-2-1型菌株B2对共生体玉米植株地下干重增加贡献明显。总体上球孢白僵菌内生定殖对玉米地下根系生物量影响大于对地上植株生物量的影响。在产卵选择性试验中,各处理组亚洲玉米螟的产卵量显著少于对照组。共生体对亚洲玉米螟产卵具有明显的趋避作用,MAT1-2-1型菌株B2对产卵的趋避作用明显,而MAT1-2-1型菌株B5的趋避作用较弱。在人工接种幼虫的试验中,处理组回收的亚洲玉米螟幼虫存活率均显著低于对照组,其中,B5组回收幼虫的存活率最低,仅为38.33%;处理组的化蛹率与对照组差异不显著,但B5组的回收幼虫化蛹率显著低于B2组和对照组,仅为34.77%,这说明MAT1-1-1型B5菌株对玉米螟幼虫发育抑制最明显。上述结果表明,不同交配型球孢白僵菌内生定殖效率有差异,在经过内生定殖后在产孢量方面有显著性提高,两个交配型菌株在联合应用时具有协同增效作用;两个交配型菌株均能够通过内生定殖与玉米形成共生体并促进玉米植株的生长,这显示球孢白僵菌和玉米之间已经建立具有互惠关系的共生体。这种共生体通过趋避亚洲玉米螟产卵、抑制幼虫存活和降低化蛹率等方面的潜力虽然不一样,但都有助于对亚洲玉米螟的可持续生态防治,也证明了共生体的建成有效提高了玉米的生态适应性,为利用球孢白僵菌内共生性实施亚洲玉米螟防控提供了新思路。     

Rapid selection of potyviral cross-protection effective mutants from the local lesion host after nitrous acid mutagenesis

Zucchini yellow mosaic virus (ZYMV) seriously damages cucurbits worldwide. Control of ZYMV by cross-protection has been practised for decades, but selecting useful mild viruses is time-consuming and laborious. Most attenuated potyviruses used for cross-protection do not induce hypersensitive reaction (HR) in Chenopodium quinoa, a local lesion host Chenopodium quinoa. Here, severe ZYMV TW-TN3 tagged with green fluorescent protein (GFP), designated ZG, was used for nitrous acid mutagenesis. From three trials, 11 mutants were identified from fluorescent spots without HR in inoculated C. quinoa leaves. Five mutants caused attenuated symptoms in squash plants. The genomic sequences of these five mutants revealed that most of the nonsynonymous changes were located in the HC-Pro gene. The replacement of individual mutated HC-Pros in the ZG backbone and an RNA silencing suppression (RSS) assay indicated that each mutated HC-Pro is defective in RSS function and responsible for reduced virulence. Four mutants provided high degrees of protection (84%–100%) against severe virus TW-TN3 in zucchini squash plants, with ZG 4-10 being selected for removal of the GFP tag. After removal of the GFP gene, Z 4-10 induced symptoms similar to ZG 4-10 and still provided 100% protection against TW-TN3 in squash, thus is considered not a genetically engineered mutant. Therefore, using a GFP reporter to select non-HR mutants of ZYMV from C. quinoa leaves is an efficient way to obtain beneficial mild viruses for cross-protection. This novel approach is being applied to other potyviruses.     

Grapevine leaf tissue colonization by the fungal entomopathogen <Emphasis Type="Italic">Beauveria bassiana</Emphasis><Emphasis Type="Italic">s.l.</Emphasis> and its effect against downy mildew

A study was conducted to examine whether Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) can colonize grapevine leaf tissues and subsequently confer protection against downy mildew caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni. Following the foliar inoculation of plants with conidial suspensions of selected B. bassiana strains, colonization of leaves by the fungus was determined using culture-based and PCR techniques at different time intervals. Seven days following B. bassiana inoculation, grapevine plants were challenged with P. viticola and symptoms were assessed by calculating the disease incidence and severity. Although all tested strains were able to colonize grapevine plants, percent colonization differed significantly among strains. Disease incidence and severity were, on the other hand, significantly reduced in B. bassiana-inoculated plants compared to control plants irrespective of strain. This study is one of very few studies investigating the promising role B. bassiana could play as a plant disease antagonist.     

Establishment of Beauveria bassiana as a fungal endophyte in potato plants and its virulence against potato tuber moth,Phthorimaea operculella (Lepidoptera: Gelechiidae)

The potato tuber moth, Phthorimaea operculella, is the most damaging potato pest in the world and is difficult to control as the larvae are internal feeders in the foliage and tubers. Entomopathogenic fungi that colonize plants as endophytes have lethal and sublethal pathological effects on insect pests. We show that Beauveria bassiana colonizes the aerial parts of potato plants endophytically after inoculation through soil drenching. Endophytic B. bassiana persisted in potato foliage for more than 50 days postinoculation. Bioassays indicated that foliage of B. bassiana-inoculated potato plants were pathogenic against larvae of P. operculella. Sublethal experiments indicated that B. bassiana negatively affected the growth, development, and reproduction of P. operculella. Development experiments showed that the weight of P. operculella pupae reared on B. bassiana-colonized potato plants (4.25 mg) was significantly less than that of those reared on uninoculated control plants (8.89 mg). Compared with newly eclosed larvae fed on control plants, those fed on B. bassiana-inoculated plants had significantly lower survivorship, with only 17.8% developing to the adult stage. Oviposition of P. operculella females reared on B. bassiana endophytically colonized plants was significantly lower (35 eggs/female) than of those reared on uninoculated plants (115 eggs/female). This study demonstrates that endophytic B. bassiana can be a potential biological control agent for the control and management of P. operculella. Comparing pupal weights of P. operculella reared on potato plants inoculated with the B. bassiana strain GZGY-1-3 and on untreated control plants, pupae from the control plants were significantly heavier than those from treated plants.     

Fungal entomopathogens as endophytes: can they promote plant growth?

Two experimental replicates were conducted to test whether strains of Beauveria brongniartii (BIPESCO2 and 2843) and Metarhizium brunneum (BIPESCO5) can endophytically colonise Vicia faba plants and improve their growth by comparing them with an endophytic strain of B. bassiana (NATURALIS^®). The plants were inoculated through foliar spray and the effect of inoculation on plant height, leaf pair number, fresh root and shoot weights was measured at 7 and 14 days post inoculation (dpi). Endophytic colonisation of different plant parts with the tested fungal strains were confirmed 7 and 14?dpi through re-isolation of inoculated fungi onto selective media and subsequent Simple Sequence Repeat (SSR) marker-based genetic identification. All tested strains were able to endophytically colonise leaves, stems, and even roots of inoculated plants 7 and 14?dpi, but per cent colonisation varied significantly among strains and plant parts within each sampling date. Foliar inoculation of plants with the tested strains increased plant height, leaf pair number, fresh shoot and root weights; however the increase was not always consistent across sampling dates in both experimental replicates. This study provides the first evidence for the endophytic colonisation of plants with two strains of B. brongniartii, an important biocontrol agent of Melolontha melolontha and other scarab beetles in several European countries, and thus extends previous reports on the ability of entomopathogenic fungi to act as endophytes. It also presents possible explanations for the lack of consistency in the plant growth promotion obtained by the foliar inoculation of entomopathogenic fungi.     

番茄内生菌的分离鉴定及菌株FQ-G3抗病促生特性

由灰葡萄孢(Botrytis cinerea)引起的灰霉病是番茄生产中最重要的病害之一,当前使用的杀菌剂因药物残留、病原菌抗药性及食品安全等原因逐渐受到限制。因此,利用拮抗微生物的生物防治逐渐成为灰霉病防控的有效策略。【目的】从番茄植株体内筛选具有抗病促生特性内生菌株并对其生防潜力进行评估,为开发番茄灰霉病生物防治新策略提供理论依据。【方法】采用组织分离法在番茄植株不同部位分离出内生细菌、真菌,结合16SrRNA和ITS序列分析,对候选菌株进行初步鉴定;通过菌株对峙培养、果实离体接种筛选对灰葡萄孢具有拮抗活性的内生菌;进一步测定菌株分泌生长素、嗜铁素的能力及其对拟南芥和番茄幼苗生长的促生特性。【结果】从番茄植株不同部位共分离出72株内生细菌和31株内生真菌,通过平板对峙法筛选出1株对多种病原菌具有较好抑菌活性的内生细菌FQ-G3,分子鉴定为Bacillus velezensis。FQ-G3对灰葡萄孢抑菌率达80.93%,并显著抑制灰葡萄孢在番茄果实上的扩展。该菌株能够分泌生长素、蛋白酶和嗜铁素,且对拟南芥、番茄幼苗具有明显的促生效果。【结论】本研究表明分离自番茄植株的内生菌FQ-G3具...     

Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)

The entomopathogenic fungus Beauveria bassiana was established in coffee seedlings after fungal spore suspensions were applied as foliar sprays, stem injections, or soil drenches. Direct injection yielded the highest post-inoculation recovery of endophytic B. bassiana. Establishment, based on percent recovery of B. bassiana, decreased as time post-inoculation increased in all treatments. Several other endophytes were isolated from the seedlings and could have negatively influenced establishment of B. bassiana. The recovery of B. bassiana from sites distant from the point of inoculation indicates that the fungus has the potential to move throughout the plant.     

<Emphasis Type="Italic">Beauveria bassiana</Emphasis> as an endophyte: a critical review on associated methodology and biocontrol potential

In the last decade there has been increased focus on the potential of endophytic Beauveria bassiana for the biocontrol of insect herbivores. Generally, detection of endophytes is acknowledged to be problematic and recovery method-dependent. Herein, we critically analyse the methodology reported for the detection of B. bassiana as endophytes following experimental inoculation. In light of the methodology, we further review the effects of endophytic B. bassiana on insect herbivores. Our review indicated the need for stringent protocols for surface sterilisation including thorough experimental controls. For molecular detection protocols by PCR, residual DNA from surface inocula must also be considered. The biocontrol potential of B. bassiana endophytes appears promising although both negative and neutral effects on insect herbivores were reported and there remains ambiguity with respect to the location and mode of action of the fungus in planta. We recommend that future studies adopt multiple techniques, including culture dependent and independent techniques for endophyte detection and elucidate the mechanisms involved against insect herbivores.     

Epidemiology of an aphid nontransmissible potyvirus in fields of nontransgenic and coat protein transgenic squash

Spread of the aphid nontransmissible Zucchini yellow mosaicvirus virus (ZYMV) strain MV was monitored over two consecutive years in field plots of nontransgenic and transgenic squash expressing the coat protein (CP) gene of the aphid transmissible strain FL of Watermelon mosaic virus (WMV). The experimental approach was to mechanically inoculate plants with ZYMV strain MV and to assess subsequent transmissions, assumed to be vectored by aphids, of this strain to nonmechanically inoculated plants. Strain MV was distinguished from other ZYMV isolates by a threonine at position 10 of the CP or by a distinct electrophoretic pattern of a Nla IV-digested genomic cDNA fragment generated by RT-PCR. ZYMV strain MV was not detected in fields of nontransgenic plants, but was apparently aphid transmitted to 77 of 3,700 plants (2%) in transgenic fields. Despite the availability of numerous test plants and conditions of high disease pressure but low selection pressure, an epidemic of ZYMV strain MV did not develop in fields of transgenic plants. In contrast, the aphid transmissible ZYMV strain NY was aphid-transmitted to 99% (446/450) of transgenic plants under similar conditions. The relevance of these results in assessing environmental risks of transgenic plants expressing CP transgenes is discussed.     

Spread of zucchini yellow mosaic potyvirus in squash in Hungary

The temporal and spatial distribution of zucchini yellow mosaic potyvirus (ZYMV) was studied in a 3000‐m² zucchini squash field. The first infected plants were found 4 weeks after the field was exposed to virus source plants. The infection increased to nearly 74% by the end of the study. Alate aphids were active from the beginning of the study and 43 species were trapped in the field. Flights of vector species Acyrthosiphon pisum and Myzus persicae peaked during the fourth week which resulted in high virus incidence 4 weeks later. There was a significant correlation between the number of vectors caught in yellow pan traps and the number of infected plants in the field. In laboratory studies evaluating 11 aphid species, Aphis pomi de Geer was identified as a new vector species of ZYMV. Although this aphid was not caught in our field studies, it may be an important contributor in other areas where cucurbits are grown in close proximity to apple or other hosts of this aphid.     

Identification of highly pathogenic Beauveria bassiana strain against Pieris rapae larvae

Seven different strains of Beauveria bassiana were used in a bioassay on Pieris rapae larvae. The results showed that an B. bassiana strain showed relatively high pathogenicity towards P. rapae larvae. The adjusted mortality rate was 92.86 %, and the infection rate was 85.71 % in 10 days post inoculation. Molecular identification was performed to identify the unknown strain. Internal Transcribed Spacer sequence analysis showed that the polymerase chain reaction amplicon length of the unknown strain of Beauveria sp. was 573 bp, and sequence similarity to the known B. bassiana sequences in the NCBI database was 99 %. The B. bassiana strain was named Bb01. The changes of proteins and PPO of P. rapae larvae infected by B. bassiana Bb01 strain at different times was determined. The activity of PPO increased in 1–6 d and decreased in 7 d again after inoculation. The B. bassiana invaded into the insect body affected the balance of the proteins and PPO.     

The Entomopathogenic Fungal Endophytes Purpureocillium lilacinum (Formerly Paecilomyces lilacinus) and Beauveria bassiana Negatively Affect Cotton Aphid Reproduction under Both Greenhouse and Field Conditions

The effects of two entomopathogenic fungal endophytes, Beauveria bassiana and Purpureocillium lilacinum (formerly Paecilomyces lilacinus), were assessed on the reproduction of cotton aphid, Aphis gossypii Glover (Homoptera:Aphididae), through in planta feeding trials. In replicate greenhouse and field trials, cotton plants (Gossypium hirsutum) were inoculated as seed treatments with two concentrations of B. bassiana or P. lilacinum conidia. Positive colonization of cotton by the endophytes was confirmed through potato dextrose agar (PDA) media plating and PCR analysis. Inoculation and colonization of cotton by either B. bassiana or P. lilacinum negatively affected aphid reproduction over periods of seven and 14 days in a series of greenhouse trials. Field trials were conducted in the summers of 2012 and 2013 in which cotton plants inoculated as seed treatments with B. bassiana and P. lilacinum were exposed to cotton aphids for 14 days. There was a significant overall effect of endophyte treatment on the number of cotton aphids per plant. Plants inoculated with B. bassiana had significantly lower numbers of aphids across both years. The number of aphids on plants inoculated with P. lilacinum exhibited a similar, but non-significant, reduction in numbers relative to control plants. We also tested the pathogenicity of both P. lilacinum and B. bassiana strains used in the experiments against cotton aphids in a survival experiment where 60% and 57% of treated aphids, respectively, died from infection over seven days versus 10% mortality among control insects. Our results demonstrate (i) the successful establishment of P. lilacinum and B. bassiana as endophytes in cotton via seed inoculation, (ii) subsequent negative effects of the presence of both target endophytes on cotton aphid reproduction using whole plant assays, and (iii) that the P. lilacinum strain used is both endophytic and pathogenic to cotton aphids. Our results illustrate the potential of using these endophytes for the biological control of aphids and other herbivores under greenhouse and field conditions.     

Comparative spatial spread overtime of Zucchini Yellow Mosaic Virus (ZYMV) and Watermelon Mosaic Virus (WMV) in fields of transgenic squash expressing the coat protein genes of ZYMV and WMV, and in fields of nontransgenic squash

The spatial and temporal patterns of aphid-vectored spread of Zucchini Yellow Mosaic Virus (ZYMV) and Watermelon Mosaic Virus (WMV) were monitored over two consecutive years in plantings of nontransgenic and transgenic squash ZW-20H (commercial cv. Freedom II) and ZW-20B, both expressing the coat protein genes of ZYMV and WMV. All test plants were surrounded by nontransgenic plants that were mechanically inoculated with ZYMV or WMV, and served as primary virus source. Across all trials, none of the transgenic plants exhibited systemic symptoms upon infection by ZYMV and WMV but a few of them developed localized chlorotic dots and/or blotches, and had low mixed infection rates [4% (6 of 139) of ZW-20H and 9% (13 of 139) of ZW-20B], as shown by ELISA. Geostatistical analysis of ELISA positive transgenic plants indicated, (i) a lack of spatial relationship on spread of ZYMV and WMV for ZW-20H with flat omnidirectional experimental semivariograms that fitted poorly theoretical models, and (ii) some extent of spatial dependence on ZYMV spread for ZW-20B with a well structured experimental semivariogram that fitted poorly theoretical models during the first but not the second growing season. In contrast, a strong spatial dependence on spread of ZYMV and WMV was found for nontransgenic plants, which developed severe systemic symptoms, had prevalent mixed infection rates (62%, 86 of 139), and well-defined omnidirectional experimental semivariograms that fitted a spherical model. Geostatistical data were sustained by virus transmission experiments with Myzus persicae in screenhouses, showing that commercial transgenic squash ZW-20H alter the dynamics of ZYMV and WMV epidemics by preventing secondary plant-to-plant spread.     

Endophytic colonisation of tobacco,corn, wheat and soybeans by the fungal entomopathogen Beauveria bassiana (Ascomycota,Hypocreales)

We demonstrate the effectiveness of three inoculation methods (foliar spray, seed immersion and root immersion) in establishing fungal the entomopathogen Beauveria bassiana as an endophyte in tobacco, corn, wheat and soybean. Colonisation of leaves by B. bassiana was assessed 7, 14, 21 and 28 days post-inoculation. There were significant differences (p < 0.001) in endophytic colonisation among the different inoculation techniques.     

Treatment of millet crop plant (Sorghum bicolor) with the entomopathogenic fungus (Beauveria bassiana) to combat infestation by the stem borer,Chilo partellus Swinhoe (Lepidoptera: Pyralidae)

Experiments were done to test if Beauveria bassiana can become an endophyte in sorghum and confer protection from stem borer. Four-week-old sorghum seedlings were treated with B. bassiana. The plants were examined for endophytic presence of B. bassiana, 30 and 60 days after treatment. Stem cultures from treated plants showed growth of B. bassiana. PCR amplification using fungal specific primers for a conserved region of β tubulin gene yielded identical 360 bp products from both B. bassiana and treated sorghum plants. In a subsequent experiment, B. bassiana treated and untreated (control) sorghum plants were artificially infested with stem borer (Chilo partellus) larvae 15 days post treatment and the extent of damage was compared. About 40% of the control plants developed dead heart while no plant in the B. bassiana treated plot did. In the surviving control plants, stem tunneling by shoot borer was significantly higher compared to B. bassiana treated sorghum plants.     

Beauveria bassiana: endophytic colonization and plant disease control

Seed application of Beauveria bassiana 11-98 resulted in endophytic colonization of tomato and cotton seedlings and protection against plant pathogenic Rhizoctonia solani and Pythium myriotylum. Both pathogens cause damping off of seedlings and root rot of older plants. The degree of disease control achieved depended upon the population density of B. bassiana conidia on seed. Using standard plating techniques onto selective medium, endophytic 11-98 was recovered from surface-sterilized roots, stems, and leaves of tomato, cotton, and snap bean seedlings grown from seed treated with B. bassiana 11-98. As the rate of conidia applied to seed increased, the proportion of plant tissues from which B. bassiana 11-98 was recovered increased. For rapid detection of B. bassiana 11-98 in cotton tissues, we developed new ITS primers that produce a PCR product for B. bassiana 11-98, but not for cotton. In cotton samples containing DNA from B. bassiana11-98, the fungus was detected at DNA ratios of 1:1000; B. bassiana 11-98 was detected also in seedlings grown from seed treated with B. bassiana 11-98. Using SEM, hyphae of B. bassiana11-98 were observed penetrating epithelial cells of cotton and ramifying through palisade parenchyma and mesophyll leaf tissues. B. bassiana11-98 induced systemic resistance in cotton against Xanthomonas axonopodis pv. malvacearum (bacterial blight). In parasitism assays, hyphae of B. bassiana 11-98 were observed coiling around hyphae of Pythium myriotylum.