Prevalence of Berkeleyomyces basicola infections in black rot-affected carrot determined using the MCM7 gene region

Thielaviopsis and related taxa are responsible for losses in several agricultural crops. In carrot these pathogens can cause black rot, mainly after harvesting. Molecular studies using species-specific DNA sequences are the principal tools for accurate identification of these pathogens. The objective of this work was to investigate which taxa are associated with black rot in carrot in Brazil using gene markers and Bayesian phylogenetic analysis. Eighteen isolates were obtained from carrots with symptoms of black rot. Through morphological characteristics all isolates were classified as Thielaviopsis-like. Bayesian inference using only MCM7 sequences and a concatenated data set (LSU, MCM7 and 60S rRNA) allowed identification of all isolates as belonging to Berkeleyomyces basicola. All isolates induced black rot in carrot. Although our studies include a small number of isolates, the results indicate the likely predominance of B. basicola in causing black rot in carrot in Brazil, and that MCM7 sequences are enough to distinguish the Berkeleyomyces species by Bayesian inference.     

Thielaviopsis basicola (Berk. et Br.) Ferraris als veroorzaker van een voor Nederland nieuwe ziekte van de tabak

Summary Tobacco black root rot, caused byThielaviopsis basicola (Berk. etBr.) Ferraris has been reported for the first time in the Netherlands. It occurred in tobacco seed beds in the Maas and Waal district, especially in the variety Kentucky Italiana.A part of the literature on this subject is briefly discussed and methods for control are given.     

Pathogenic fungi involved in root rot of peas in the Netherlands and their physiological specialization

Research on root rot pathogens of peas in the Netherlands has confirmed the prevalence ofFusarium solani, F. oxysporum, Pythium spp.,Mycosphaerella pinodes andPhoma medicaginis var.pinodella. Aphanomyces euteiches andThielaviopsis basicola were identified for the first time as pea pathogens in the Netherlands. Other pathogens such asRhizoctonia solani andCylindrocarpon destructans were also found on diseased parts of roots. F. solani existed in different degrees of pathogenicity, and was sometimes highly specific to pea, dwarf bean of field bean, depending on the cropping history of the field.A. euteiches was specific to peas, whereasT. basicola showed some degree of physiological specialization.     

Characterization of isolates of binucleate Rhizoctonia spp. associated with strawberry black root rot complex using fatty acid methyl ester (FAME) profiles

Black root rot is an important disease of strawberry caused by a complex of fungi that includes species of Rhizoctonia. In this study, a modified MIDI method (Microbial Identification System) was investigated for its utility to differentiate isolates of the three different anastomosis groups (AGs) of binucleate Rhizoctonia spp., associated with strawberry black root rot complex representing AG-A, AG-G, and AG-I. A total of 11 fatty acids were detected, and the FAME profiles for isolates of the three different AGs of Rhizoctonia spp. varied quantitatively and qualitatively. Moreover, the modified MIDI method will be a useful discriminatory tool for fungal identification and classification of the AGs of binucleate Rhizoctonia spp. associated with strawberry black root rot complex.     

117个玉米品种对禾谷镰孢根腐病的抗性鉴定及根腐病对苗势与产量的影响

为评价东北地区玉米主推品种对禾谷镰孢根腐病的抗性水平,探究根腐病发生与苗势、产量损失间的关系,采用人工接种方法鉴定东北地区117个玉米主推品种对禾谷镰孢根腐病的抗性水平,应用室内盆栽试验分析自交系LN810在施用氮、磷、钾及复合肥后对禾谷镰孢根腐病发生和苗势的影响,在田间对先玉335接种禾谷镰孢菌Fusarium graminearum后探究根腐病发生程度对其苗势及产量的影响。结果显示,117个玉米品种对禾谷镰孢根腐病的抗性差异明显,鉴定出高抗品种14个,抗病品种44个,中抗品种47个,感病品种12个,其中表现中抗以上的品种占89.74%,且中早熟材料均表现为抗性。与未施肥相比,施用磷钾肥后玉米禾谷镰孢根腐病发生率最低,为22.03%。按照禾谷镰孢根腐病发生程度从轻到重将先玉335群体划分为一、二、三类苗,级别越高苗势越弱,产量测定发现二、三类苗的平均产量较一类苗的平均产量分别下降了39.97%和76.39%。表明东北地区主推玉米品种大部分对禾谷镰孢根腐病表现出抗性,但仍有部分品种存在较大感病风险,且该病害的发生程度与幼苗长势和产量呈负相关,施用磷钾肥可降低该病害的发生率。     

Interaction between root rot basidiomycetes and Phytophthora species on pedunculate oak

Forest declines are usually complex multifactorial phenomena that involve interactions between different factors. The possible interaction between different types of mycelial pathogens was investigated through artificial inoculation of oak seedlings, involving two root rot basidiomycetes, Collybia fusipes and Armillaria mellea, and two Phytophthora species, P. cinnamomi and P. cambivora. These pathogens were inoculated onto young Quercus robur saplings in greenhouse conditions, either alone or combining a root rot basidiomycete with a Phytophthora species. Three out of the four Phytophthora spp.*root rot basidiomycete combinations tested resulted in significantly greater damage to the oak host than the sum of the damages induced by the individual pathogens. This positive interaction could be significant in oak decline syndrome.     

Identification and pathogenicity of Botryosphaeriaceae species associated with root and stem rot of sweet potato in Brazil

The sweet potato (Ipomoea batatas) is characterized by the production of tuberous roots rich in starch and is one of the most produced and consumed vegetables in Brazil. Botryosphaeriaceae, among other fungi, are known to cause root and stem rot of sweet potato. However, no representative and accurate study has been performed for the correct identification of these fungal species in sweet potato in Brazil. Therefore, this study aimed to identify the Botryosphaeriaceae species associated with root and stem rot of sweet potato and confirm their pathogenicity. Tuberous roots and stems of sweet potato with rot symptoms were collected in production fields and markets and used for fungal isolations. The identification of fungi was based on the morphology of reproductive structures and phylogenetic analyses of the gene regions ITS, tef1-α, and rpb2. The following species were identified: Lasiodiplodia theobromae, L. hormozganensis, Macrophomina phaseolina, M. euphorbiicola, M. pseudophaseolina, and Neoscytalidium dimidiatum. For the pathogenicity test, one representative isolate for each species was inoculated in healthy tuberous roots and in 30-day-old healthy seedlings. Black and necrotic lesions on tuberous roots and stems were observed in all replications and resulted in the death of some seedlings. This is the first report of L. hormozganensis, M. pseudophaseolina, and M. euphorbiicola, as causal agents of the stem and root rot of sweet potato and N. dimidiatum as a causal agent of stem rot worldwide.     

Phytophthora rot of alpine delphinium caused by Phytophthora sp. kelmania

Severe stunting and root rot were observed on alpine delphinium plants (Delphinium elatum) from Aomori Prefecture, Japan, in 2010 and 2011. A Phytophthora isolate from the diseased crown was identified as Phytophthora sp. kelmania based on morphological characteristics and DNA sequence data. Inoculation of alpine delphinium plants with the isolate produced a similar root rot. Pathogenicity of the isolate on four species of plants that are known hosts for P. sp. kelmania was confirmed. We propose the name “Phytophthora rot” (eki-byo in Japanese) for the present new disease on alpine delphinium.

     

Root‐knot nematodes do not affect black root rot disease severity on watermelon and bottle gourd

The effects of root‐knot nematodes on black root rot of watermelon and bottle gourd were studied using field surveys and co‐inoculation tests with Meloidogyne incognita (southern root‐knot nematode) and Diaporthe sclerotioides. The results of the field survey suggested that root‐knot nematodes had little effect on either the severity of black root rot or infection with D. sclerotioides. None of the three fields showed a significant positive correlation between disease severity and nematode gall index, with low correlation coefficients. Co‐inoculation experiments under controlled conditions found no significant effect of root‐knot nematodes on black root rot of watermelon and bottle gourd based on area under disease progress curves (AUDPC). These results were supported by the quantities of DNA of the two agents in root tissues because no significant difference was found between dual‐ and single‐inoculation treatments with M. incognita and/or D. sclerotioides. These findings suggest that root‐knot nematodes probably do not affect the infection of watermelon or bottle gourd roots by D. sclerotioides or the incidence of black root rot in these crops caused by this fungus.     

Black root rot of cucurbits caused by Phomopsis sclerotioides in Japan and phylogenetic grouping of the pathogen

Although the causal agent of black root rot of Cucurbitaceae in Japan has been proposed as Phomopsis sclerotioides, the species identification of the pathogen has remained inconclusive because of a lack of spore formation. We confirmed that a Japanese isolate of Phomopsis sp. obtained from a diseased pumpkin root produced pycnidia containing α spores in sterilized bean pods. In phylogenetic analyses of rDNA-ITS regions, nine Japanese Phomopsis sp. isolates from melon, watermelon grafted onto bottle gourd, and pumpkin diagnosed with black root rot, formed a single clade with P. sclerotioides standard isolates. We identified the causal agent of the black root rot of melon, pumpkin, bottle gourd, and watermelon in Japan as P. sclerotioides and propose the Japanese name “Phomopsis-negusare-byo” for the disease. Patterns of random amplified polymorphic DNA (RAPD) of these Japanese isolates were also similar to those of P. sclerotioides, thus supporting the species identification. However, mycelial incompatibilities were found for many combinations among these P. sclerotioides isolates, suggesting some genotypic variations of this fungus in Japan at a level that the RAPD analyses cannot discriminate. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB201430 to AB201444     

Comparison of the Susceptibility of Quercus petraea, Q. robur and Q. rubra to Collybia fusipes

Collybia fusipes is the cause of a root rot of Quercus petraea (sessile oak), Q. robur (pedunculate oak) and Q. rubra (red oak). This parasite is often reported to cause problems in stands of red oaks and field evidence suggests that this North American oak species is more susceptible than the two European oak species. Young saplings of the three oak species and of chestnut, Castanea sativa, in the glasshouse, and also mature sessile and red oaks growing in the same stand were inoculated with C. fusipes to compare their susceptibility. Red oak, both as young seedlings and mature trees, was more susceptible to C. fusipes than sessile oak. Chestnut seedlings were as susceptible as sessile oak. Susceptibility of pedunculate oak seedlings was intermediate between red oak and sessile oak. In one experiment this species was significantly less susceptible than red oak, and in the other it was as susceptible.     

Interactions between the biocontrol agent Pseudomonas fluorescens CHA0 and Thielaviopsis basicola in tobacco roots observed by immunofluorescence microscopy

Pseudomonas fluorescens CHA0 protects plants from damage caused by several soilborne fungi. In this work, immunofluorescence microscopy was used to investigate the colonization of tobacco roots by CHA0 and its physical relationship with the black root rot fungus Thielaviopsis basicola . The pseudomonad colonized the rhizoplane shortly after planting of tobacco seedlings in sterile soil microcosms, in which it had been introduced as soil inoculant. CHA0 was found between and inside cells in the epidermis and the cortex, as well as in the xylem vessels, within 4–7 days after planting of seedlings. The presence of CHA0 delayed the colonization of the interior of tobacco roots by T. basicola compared with the treatment in which only the fungus had been inoculated. Likewise, the pseudomonad reduced the extent of black root rot from 82% to 28%. However, CHA0 was seldom found in contact with the mycelium of T. basicola or in its vicinity, indicating that direct colonization of the mycelium of T. basicola by CHA0 was not required for protection of tobacco against black root rot. Overall, the results suggest that the interior of the root is a key site for implementation of the strain's biocontrol activity against soilborne plant-pathogenic fungi.     

冠菌素诱导甘蓝幼苗抗黑腐病及其机理初步研究

以甘蓝 Brassica oleracea L.感病品种"庆丰"为材料,通过叶面喷施冠菌素(coronatine,COR)和人工接种黑腐病菌——野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris)的方法,研究了COR诱导甘蓝幼苗抗黑腐病的效果及其生理生化机制。结果表明, COR在质量浓度为0.01、0.1和1.0 mg/L下均具有诱导甘蓝幼苗抗黑腐病的效果,其中1.0 mg/L 处理的病情指数为38.2,诱抗率为43.7%,诱导抗病效果最好。与对照相比,随着COR质量浓度的提高,叶片中过氧化氢酶(CAT)活性和超氧阴离子(O2-)产生的速率明显增加,而过氧化物酶(POD)的活性增加不明显,超氧化物歧化酶(SOD)活性和丙二醛(MDA)的含量明显下降。表明COR具有诱导甘蓝幼苗抗黑腐病的作用,而这种作用可能与COR能调控甘蓝幼苗中抗氧化酶活性、提高O2-产生速率以及降低MDA的含量相关。     

Nonpathogenic Binucleate Rhizoctonia spp. and Benzothiadiazole Protect Cotton Seedlings Against Rhizoctonia Damping-Off and Alternaria Leaf Spot in Cotton

ABSTRACT Recent reports have shown induction of resistance to Rhizoctonia root rot using nonpathogenic strains of binucleate Rhizoctonia spp. (np-BNR). This study evaluates the biocontrol ability of several np-BNR isolates against root and foliar diseases of cotton in greenhouse trials, provides evidence for induced systemic resistance (ISR) as a mechanism in this biocontrol, and compares the disease control provided by np-BNR with that provided by the chemical inducer benzothiadiazole (BTH). Pretreatment of cotton seedlings with np-BNR isolates provided good protection against pre- and post-emergence damping-off caused by a virulent strain of Rhizoctonia solani (AG-4). Seedling stand of protected cotton was significantly higher (P < 0.05) than that of nonprotected seedlings. Several np-BNR isolates significantly reduced disease severity. The combination of BTH and np-BNR provided significant protection against seedling rot and leaf spot in cotton; however, the degree of disease reduction was comparable to that obtained with np-BNR treatment alone. Significant reduction in leaf spot symptoms caused by Alternaria macrospora occurred on cotyledons pretreated with np-BNR or sprayed with BTH, and the np- BNR-treated seedlings had significantly less leaf spot than BTH-treated seedlings. The results demonstrate that np-BNR isolates can protect cotton from infections caused by both root and leaf pathogens and that disease control was superior to that observed with a chemical inducer.     

Effect of chitin on biological control activity of Bacillus spp. and Trichoderma harzianum against root rot disease in pepper (Capsicum annuum) plants

Two bacterial isolates and one strain of Trichoderma harzianum were tested alone and in combination with chitin for efficacy in control of root rot disease caused by Phytophthora capsici and Rhizoctonia solani in pepper plants under greenhouse conditions. These bacteria (Bacillus subtilis HS93 and B. licheniformis LS674) were isolated from repeatedly washed roots of pepper plants. In in vitro assays, HS93, LS674 and T. harzianum were antagonistic against P. capsici and R. solani and produced high levels of chitinase. Seed treatment and root drenching with bacterial suspensions of HS93 with 0.5% chitin was more effective against Phytophthora and Rhizoctonia root rot than addition of the organisms without chitin. LS674 and T. harzianum reduced Rhizoctonia but not Phytophthora root rot. In two greenhouse tests, seed treatment and root drenching with HS93 amended with chitin enhanced its biocontrol activity against P. capsici but not on R. solani. The effects of LS674 and T. harzianum against R. solani were significantly enhanced when they were used as suspensions with 0.5% chitin for root drenching, but this had no effect on P. capsici. In both greenhouse experiments, the use of 0.5% chitin alone for root drenching reduced Rhizoctonia root rot. Reduction of root rot disease was accompanied by increased yield. These results show that the antagonistic activity of HS93, LS674 and T. harzianum may be stimulated by chitin resulting in significant improvements in their effectiveness against pathogens.     

Pyriproxyfen,a novel insect growth regulator for controlling whiteflies: Mechanisms and resistance management

Pyriproxyfen, a novel juvenile hormone mimic, is a potent suppressor of embryogenesis and adult formation of the sweetpotato whitefly, Bemisia tabaci (Gennadius), and the greenhouse whitefly, Trialeurodes vaporariorum (Westwood). Dipping of cotton or tomato seedlings infested with 0 to 1-day-old eggs in 0.1 mg litre^?1 resulted in over 90% suppression of egg hatch of both B. tabaci and T. vaporariorum. Older eggs were affected to a lesser extent. Exposure of whitefly females to cotton or tomato seedlings treated with pyriproxyfen resulted in oviposition of non-viable eggs. The LC90 values for egg viability of B. tabaci and T. vaporariorum exposed to treated plants were 0.05 and 0.2 mg litre^?1, respectively. Treatment of whitefly larvae with 0.04–5 mg litre^?1 resulted in normal development until the pupal stage; however, adult emergence was totally suppressed. Second instars of B. tabaci exposed to 5 mg litre^?1 pyriproxyfen, excreted honeydew at a level similar to the control level until the fourth instar (pupation), after which a strong reduction was observed. Inhibition of egg-hatch on the lower surface of cotton leaves was observed when their upper surface was treated with 1–25 mg litre^?1, indicating a pronounced translaminar effect. These findings indicate that pyriproxyfen is an efficient control agent of both B. tabaci and T. vaporariorum. The compound has been used successfully for controlling whiteflies in Israeli cotton fields since 1991. Adults of B. tabaci collected from a rose greenhouse and from adjacent cotton fields were monitored during 1991–1993 for their susceptibility to pyriproxyfen. A high level of resistance was recorded in whiteflies collected from a greenhouse after three successive applications of pyriproxyfen. Based on LC50 values, the resistance ratio for egg-hatch suppression was 554-fold and, for adult emergence failure, 10-fold. However, a single treatment of pyriproxyfen in cotton fields during the summer season (according to an insecticide resistance management (IRM) strategy) did not alter appreciably the susceptibility of B. tabaci to this compound. In order to prevent development of resistance, an attempt should be made to restrict its use to one treatment per crop season applied during the peak activity of the pest. Pyriproxyfen can be alternated with other novel compounds such as buprofezin and diafenthiuron for controlling whiteflies in cotton, vegetables and ornamentals as part of integrated pest management (IPM) and IRM strategies. In pyriproxyfen- or buprofezin-resistant strains of B. tabaci or T. vaporariorum, no appreciable cross-resistance was observed among pyriproxyfen, buprofezin and diafenthiuron.     

Molecular identification and pathogenicity of Fusarium and Alternaria species associated with root rot disease of wolfberry in Gansu and Ningxia provinces,China

Wolfberry (Lycium barbarum) ranks in the top 10 best-selling medicinal plants in China and it has been used for centuries as a medicine and a food supplement. It is suggested to have benefits on human health due to the rich content of polysaccharides, carotenoids, flavonoids, and alkaloids contained in its fruits, leaves, and root bark. Recently, severe root rot diseases have been causing plant losses in major growing areas. Here, we report fungi causing root rot disease in Chinese wolfberry plants. The analysis of nucleotide sequences of the internal transcribed spacer (ITS) region revealed a total of 92 isolates isolated from both soil and plant material samples. Fusarium spp. were the most abundant (58%), followed by Penicillium spp. (9%), and Alternaria spp. (5%). Fusarium spp. included F. oxysporum (36%), F. solani (30%), F. chlamydosporum (9%), F. nematophilum (9%), and F. tricinctum (8%). Sequences from the translation elongation factor 1-α gene (TEF-1α) were used to confirm the identity of Fusarium spp. and showed the predominance of F. oxysporum and F. solani. To confirm the pathogenicity of isolates, four isolates belonging to Fusarium spp. and one isolate belonging to Alternaria spp., isolated from wolfberry root tissues with root rot symptoms, were tested in outdoor and laboratory conditions. Results revealed that the five tested isolates were pathogenic with varying degrees of aggressiveness and ability to induce symptoms of root rot in wolfberry seedlings. The five isolates were recovered from inoculated seedlings, completing Koch's postulates. This is the first report on causative agents of root rot in Chinese wolfberry.