Identification and characterization of Phoma tracheiphila mutants impaired in pathogenicity following Agrobacterium-mediated mutagenesis

Mal secco disease, caused by the pathogenic fungus Phoma tracheiphila, is a devastating disease of susceptible citrus species, especially lemon. To study the molecular interactions between the pathogen and its host, a method for identifying the genes involved in pathogenicity is needed. This work describes the transformation of P. tracheiphila phialoconidia by Agrobacterium tumefaciens, and the generation of mutated P. tracheiphila isolates exhibiting reduced virulence on rough lemon seedlings. A rapid, replicable, and reliable method for screening P. tracheiphila mutants to assess their virulence by using rough lemon seedlings was developed. Among 2263 transformants obtained, three were non-virulent and 43 displayed reduced virulence. In addition, one of the transformants, which exhibited virulence similar to that of the wild type, was used for in planta visualization of the fungus progression through the plant xylem. To our knowledge, this is the first report of A. tumefaciens-mediated transformation of P. tracheiphila, and subsequent screening of the transformants to identify non-virulent mutants.     

Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

 Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O₂ ⁻, diaminobenzidine (DAB) methods for microscopic detection of H₂O₂, and cerium chloride methods for ultrastructural detection of H₂O₂. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility. Received: June 3, 2002 / Accepted: July 31, 2002     

The effect of hydrogen peroxide on the viability of tomato cells and of the fungal pathogen Cladosporium fulvum

An oxidative burst was previously demonstrated to be induced in tomato plants by race specific elicitors of the fungal pathogen Cladosporium fulvum . The in planta levels of H₂O₂estimated to occur during elicitor treatment, were compared with the levels required to show toxicity to host cells and to the fungal pathogen. Injection of Cf-9 tomato leaves with 100 m m H₂O₂caused an insignificant degree of necrosis and 1m H₂O₂was required to cause complete leaf necrosis comparable to that induced by the AVR9 elicitor. Assays with Cf-5 tomato cell suspensions confirmed the low toxicity of H₂O₂to tomato cells but, as expected, the addition of Fe²⁺with H₂O₂(or with intercellular fluids containing AVR5 elicitor) enhanced cell death as determined by the Evans Blue assay. Germination and germ tube growth of conidia of C. fulvum were significantly retarded by 4–5 m m H₂O₂, and at higher concentrations, death of germ tubes was observed (ED₅₀=22 m m), as determined by the fluorescein diacetate assay. The addition of Fe²⁺with H₂O₂had little effect on fungal growth or viability in vitro . These results suggest that the amount of H₂O₂accumulating during an elicitor-induced response in leaves may be sufficient to affect fungal colonization but not to affect viability of host cells unless the Fe²⁺status in the apoplast is in some way altered by the elicitor to facilitate OH^.production via the Fenton reaction.     

Sclerotinia sclerotiorum catalase SCAT1 affects oxidative stress tolerance,regulates ergosterol levels and controls pathogenic development

Reactive oxygen species (ROS) are essential for pathogenic development of Sclerotinia sclerotiorum. A key question for S. sclerotiorum and many other pathogens concerns how fungi tolerate/dampen the oxidative environment during growth and pathogenesis. Regulatory components of oxidative stress include both enzymatic and non-enzymatic antioxidants. Catalases are a ubiquitous family of enzymes that play an important role in the enzymatic detoxification of ROS by converting hydrogen peroxide (H₂O₂) to water and molecular oxygen. The genome of the omnivorous pathogen S. sclerotiorum contains seven predicted catalase genes. In this study we evaluate and functionally characterize the type A catalase (Scat1) in S. sclerotiorum, whose expression is highly induced during host infection. Insertional inactivation of Scat1 (ΔScat1) resulted in hyperbranching of hyphae accompanied by slower growth and smaller sclerotia. ΔScat1 strains were attenuated in pathogenicity and rendered the fungus hypersensitive to Sodium dodecyl sulfate (SDS), as well as to osmotic and salt stresses. Unexpectedly, ΔScat1 exhibited increased tolerance to H₂O₂, suggesting that although a member of the catalase family, generally associated with amelioration of oxidative stress, Scat1 is probably not required for detoxification of this oxygen species and presumably has different function(s). ΔScat1 strains had a 2-fold decrease in ergosterol content, and overall lower sterol levels compared to the wild-type strain. These observations are consistent with increased resistance to the polyene drugs amphotericin-B and nystatin. Taken together, our results suggest Scat1 is involved in modulation of ROS in a manner that deviates from the detoxification of H₂O₂, alters membrane integrity and contributes to the pathogenic success of S. sclerotiorum.     

Evolution of pathogenicity controlled by small,dispensable chromosomes in Alternaria alternata pathogens

Alternaria alternata includes seven pathogenic variants, called pathotypes, which produce host-selective toxins (HSTs) as determinant factors for pathogenicity. The gene clusters for HST biosynthesis were identified from six pathotypes (Japanese pear, strawberry, tangerine, apple, tomato and rough lemon) and were found to reside on small chromosomes of <2.0 Mb in most strains tested. We isolated mutants lacking the small chromosomes from the strawberry, apple and tomato pathotypes and showed that the small chromosomes are dispensable for growth. In this review, we summarize our current understanding of the evolution of pathogenicity controlled by small, dispensable chromosomes in Alternaria alternata pathogens.     

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H₂O₂ generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H₂O₂ generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.     

Roles of reactive oxygen species in interactions between plants and pathogens

The production of reactive oxygen species (ROS) by the consumption of molecular oxygen during host–pathogen interactions is termed the oxidative burst. The most important ROS are singlet oxygen (¹O₂), the hydroxyperoxyl radical (HO₂·), the superoxide anion , hydrogen peroxide (H₂O₂), the hydroxyl radical (OH^-) and the closely related reactive nitrogen species, nitric oxide (NO). These ROS are highly reactive, and therefore toxic, and participate in several important processes related to defence and infection. Furthermore, ROS also play important roles in plant biology both as toxic by-products of aerobic metabolism and as key regulators of growth, development and defence pathways. In this review, we will assess the different roles of ROS in host–pathogen interactions with special emphasis on fungal and Oomycete pathogens.     

Sequence and evolutionary analysis of ribosomal DNA from Huanglongbing (HLB) isolates of Western India

Citrus greening (Huanglongbing, HLB) is a widespread and economically important citrus disease all over the world. The disease is caused by a phloem-limited fastidious gram negative bacterium, “Candidatus Liberibacter spp.” which belongs to the alpha-proteobacteria group classified on the basis of its 16SrDNA sequence. Although the pathogen has been classified under three distinct groups, viz. Asian, African and American isolates, nothing is known about the status and the molecular variabilities among the Indian HLB isolates collected from different citrus cultivars grown in India. Five different HLB isolates showing variable symptoms based on their severity of infection on different citrus, viz. Mosambi, Rangpur lime, Cleopatra mandarin, acid lime and rough lemon, were studied by PCR amplification, sequence and evolutionary analysis of their 16S and 16S/23S rDNA intergenic regions. Analysis of the 16S/23S rDNA intergenic region separated all five Indian isolates from existing African isolates but failed to differentiate among Asian, American and Indian isolates. However, further analysis of complete 16S rDNA clearly indicated that Indian isolates fall within the Asian HLB group. Overall, our results suggest that all the five Indian HLB isolates taken for the current analysis belong to the Candidatus Liberibacter asiaticus strain, which showed distinct sequence variabilities and produced noticeable symptoms on the citrus trees. These results provide a robust framework for understanding how differences in pathogenicity among various HLB isolates may be related to evolutionary history.     

Chlorocholine chloride mediated resistance mechanism and protection against leaf spot disease of Stevia rebaudiana Bertoni

Investigations have been carried out to determine the influence of chlorocholine chloride on induction of the resistance mechanisms of Stevia rebaudiana against leaf spot disease, caused by Alternaria alternata. The paper also focuses an impact of chlorocholine chloride induced resistance on reduction of leaf spot disease. Chlorocholine chloride is attributed to its significant role in defence responses through augmentation of phenol and salicylic acid content as well as stimulation of phenylalanine ammonia lyase and peroxidase activity in S. rebaudiana following inoculation with A. alternata. Histochemical studies revealed that fungal invasion as well as infection process was appreciably reduced in chlorocholine chloride treated plants through peroxidase-H₂O₂ mediated strengthening of cell wall. The overall study highlights the significant role of chlorocholine chloride in induction of resistance in S. rebaudiana against A. alternata.     

Soybean plants expressing an active oligomeric oxalate oxidase from the wheat gf-2.8 (germin) gene are resistant to the oxalate-secreting pathogen Sclerotina sclerotiorum

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (Lib.) De Bary is a serious fungal disease of soybean. Senescing petals provide a starting nutrient source for the invasion of healthy tissue by the advancing oxalic acid secreting fungal hyphae. Since oxalic acid is a major pathogenicity factor of SSR, transgenic soybean capable of degrading oxalic acid may be resistant to the pathogen. Transgenic soybean plants were produced byAgrobacterium -mediated transformation with the wheat germin gene (gf-2.8) encoding an oligomeric protein, oxalate oxidase (OxO), which oxidizes oxalic acid to carbon dioxide and hydrogen peroxide (H₂O₂). Transgenic soybean homozygous for 35S- gf-2.8 produced an approx. 130 kDa protein indistinguishable from wheat germin, and with OxO activity. OxO activity was prominent in cell walls proximal to the site of pathogen attack. The transgenics had greatly reduced disease progression and lesion length following cotyledon and stem inoculation with S. sclerotiorum indicating that the germin gene product conferred resistance to SSR. This is the first report of plant resistance to the fungal pathogen S. sclerotiorum in transgenic plants expressing OxO.     

Overproduction of reactive oxygen species involved in the pathogenicity of Fusarium in potato tubers

Differences in virulence between Fusarium sulphureum and Fusarium sambucinum were compared. Changes in reactive oxygen species production and metabolism in inoculated slices of potato tubers were also compared. The result showed that Fusarium infection induced significant production of ROS, lipid peroxidation and loss of cell membrane integrity, but low activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Compared to F. sambucinum, F. sulphureum led larger lesion diameters on potato tubers and slices. It resulted in more superoxide anion ($O_2^{-}$) and earlier peak of hydrogen peroxide (H₂O₂), but lower activity of catalase (CAT) and APX, and accompanied with higher malondialdehyde (MDA) content and lower cell membrane integrity. These findings suggested that overproduction of ROS involved in the pathogenicity of Fusarium in potato tubers.     

Ultrastructural Localization of Hydrogen Peroxide in Host Leaves Treated with AK-toxin I Produced by Alternaria alternata Japanese Pear Pathotype

The rapid generation of reactive oxygen species (ROS), called the oxidative burst, is one of the earliest host responses to pathogen infection or elicitor treatments. Therefore, we looked for the induction of ROS generation in Japanese pear leaves by the host-specific toxin, AK-toxin I using a cytochemical method for detecting H₂O₂. A small amount of non-specific generation of H₂O₂ was found in the cell walls in toxin- and water-treated susceptible and resistant leaves. Thus, the generation of H₂O₂ at cell walls appears to be caused by wounding stress during sampling. Specific generation of ROS, however, was found only in the membrane fragments and extended desmotubules characteristic of modified sites of the plasma membrane in the toxin-treated susceptible leaves. In addition, generation of H₂O₂ at plasma membranes was observed with higher frequency in toxin-treated susceptible leaves. This result indicates that the H₂O₂ generation was associated with damaged sites in the plasmalemma after toxin treatment and perhaps with the formation of membrane fragments from altered portions of the invaginated plasma membrane. Received 21 September 2001/ Accepted in revised form 25 October 2001     

Polygalacturonase S31PG1 from <Emphasis Type="Italic">Geotrichum candidum</Emphasis> citrus race S31 expressed in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> versus S31PG2 regarding soft rot on lemon fruit

Gene S31pg1, which encodes a polygalacturonase (PG), was previously isolated from citrus race S31 of Geotrichum candidum, the causal agent of citrus sour rot. We have now isolated and sequenced an additional PG gene, S31pg2, with 95% identity to S31pg1 in the mature proteins. To evaluate the contribution of the two PG genes in the development of citrus sour rot, each gene was expressed in the fission yeast Schizosaccharomyces pombe. Both genes conferred PG activity to the yeast. Crude enzyme solutions containing S31PG1 severely degraded the albedo tissue of lemon peel, but those containing S31PG2 did not. Concentrated crude S31PG1 solutions also caused soft rot on lemon fruit, indicating that not S31PG2 but S31PG1 is an important pathogenicity factor in citrus sour rot. Next, the protopectinase (PP) activity of each PG was measured. Although S31PG1 and S31PG2 are highly homologous, S31PG1 had high PP activity, whereas S31PG2 had much lower activity. PG from G. candidum noncitrus race S63 (nonpathogenic to citrus fruits) was also assayed but did not have any PP activity at all. These results suggest that the different PP activities of the PGs are a key to the pathogenicity of G. candidum to lemon fruit.     

福建省柑橘褐斑病菌的分离、鉴定和系统进化分析

为明确在福建省南平市的橘柚和三明市的温州蜜橘上发现的疑似柑橘褐斑病的病原菌种类,采用组织分离法获得纯化菌株,通过回接法验证菌株的致病性,利用形态学特征对病原菌进行初步鉴定,并采用最大似然法以多聚半乳糖醛酸酶基因endoPG为靶标对本研究以及国内外已报道的链格孢菌株构建系统发育树,分析其遗传多样性。结果表明,从病组织中共分离获得26株纯培养菌株,经形态学鉴定均为链格孢菌Alternaria spp.。利用分生孢子液接种橘柚离体嫩叶发现,有22株菌株能侵染橘柚叶片并产生与田间相似的褐斑病症状,确认该病害为链格孢引起的柑橘褐斑病。系统发育树分析结果显示,分离所得的26株菌株均聚在已报道的4个柑橘链格孢进化分支Clade1～Clade4中,其中21株菌株聚在国内特有的分支Clade4中,有3株菌株和1株菌株分别聚在国内外兼有的分支Clade3和Clade1中,1株菌株聚在国外特有的分支Clade2中,表明在福建省采集的这些柑橘褐斑病菌均为链格孢菌,且遗传多样性较丰富。     

Effect of riboflavin on postharvest disease of Asia pear and the possible mechanisms involved

The effects of riboflavin (vitamin B₂) against Alternaria rot caused by Alternaria alternata and its possible mechanism in harvested Zaosu pear fruit were investigated. Riboflavin at a concentration of 1.0?mM effectively inhibited development of Alternaria rot and enhanced the activities of defense-related enzymes, such as phenylalanine ammonia-lyase (PAL), polyphenoloxidase (PPO) and peroxidase (POD), and increased accumulation of flavonoids, phenolics and lignin. Riboflavin also affected reactive oxygen metabolism of pear fruit by increasing O ₂ ^?C production and H₂O₂ content, and enhancing the activities of the main detoxifying enzymes, such as catalase (CAT) and superoxide dismutase (SOD). Spore germination and mycelial growth of A. alternata were inhibited by riboflavin and the inhibitory effect was highly correlated with the riboflavin concentration used in this study. It is suggested that the effects of riboflavin on Alternaria rot in pear fruit may be associated with its direct fungitoxic property against the pathogens, and the elicitation of biochemical defense responses in the fruit.     

BTH处理对甜瓜叶片活性氧代谢的诱导作用

为了明确活性氧（reactive oxygen species,ROS）代谢在甜瓜抗病性诱导中的作用,以抗白粉病甜瓜品种Tam Dew和感病品种卡拉克赛幼苗为材料,通过盆栽试验研究了苯丙噻二唑（BTH）喷雾或白粉菌接种后甜瓜叶片超氧阴离子（O2.-）产生速率、过氧化氢（H2O2）含量及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、苯丙氨酸解氨酶（PAL）活性的变化。BTH处理或白粉菌接种均可诱导甜瓜叶片SOD、PAL活性升高,抑制CAT活性,导致叶组织O2.-产生速率和H2O2含量增加,BTH喷雾＋白粉菌接种比二者单独处理效果更好。结果表明,BTH处理后叶片O2.-产生速率提高和H2O2积累是甜瓜抗白粉病能力提高的重要机制,BTH通过诱导ROS代谢酶活性调节H2O2含量,且BTH诱导的甜瓜抗病性与品种的基础抗性有关。     

Histochemical studies on the accumulation of reactive oxygen species (O2− and H2O2) in the incompatible and compatible interaction of wheat—Puccinia striiformis f. sp. tritici

The generation and accumulation of reactive oxygen species (ROS), superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂), were studied in the interaction between wheat cv. ‘Suwon 11’ and two races of Puccinia striiformis f. sp. tritici (avirulent and virulent). Generation of O₂⁻ and H₂O₂ was analyzed histochemically using nitroblue tetrazolium (NBT) and 3,3-diamino-benzidine (DAB), respectively. At the pre-penetration stage during appressorium formation both stripe rust races induced H₂O₂ accumulation in guard cells. In the incompatible interaction, a rapid increase of O₂⁻ and H₂O₂ generation at infection sites was detected. The percentage of infection sites showing NBT and DAB staining was 36.1% and 40.0%, respectively, 12 h after inoculation (hai). At extended incubation time until 24 hai, percentage of infection sites showing H₂O₂ accumulation further increased, whereas those exhibiting O₂⁻ accumulation declined. The early infection stage from 12 to 24 hai coincided with primary haustoria formation in mesophyll cells. In contrast, in the compatible interaction, O₂⁻ and H₂O₂ generation could not be detected in most of the infection sites. In the incompatible interaction, intensive DAB staining was also determined in mesophyll cells, especially in cell walls, surrounding the infected cells 16–24 hai; thereafter, these cells contained fluorescing compounds and underwent hypersensitive response (HR). The number of necrotic host cells surrounding the infection sites increased continuously from 20 to 96 hai. It might be concluded that H₂O₂ accumulation during the early infection stage is associated with the occurrence of hypersensitive cell death and that resistance response is leading to arrest the avirulent race of the obligate stripe rust pathogen. In the compatible interaction at 96 hai, H₂O₂ accumulation was observed in mesophyll cells surrounding the rust lesion.     

<Emphasis Type="Italic">Alternaria alternata</Emphasis> apple pathotype (<Emphasis Type="Italic">A. mali</Emphasis>) causes black spot of European pear

A disease caused by Alternaria alternata occurred on the leaves of European pear cultivar Le Lectier in Niigata Prefecture, Japan, and was named black spot of European pear. In conidial inoculation tests, the causal pathogen induced not only small black lesions on the leaves of European pear cultivar Le Lectier, but severe lesions on the leaves of apple cultivar Red Gold, which is susceptible to the A. alternata apple pathotype (previously called A. mali) causing Alternaria blotch of apple. Interestingly, the apple pathotype isolate showed the same pathogenicity as the European pear pathogen. HPLC analysis of the culture filtrates revealed that A. alternata causing black spot of European pear produced AM-toxin I, known as a host-specific toxin of the A. alternata apple pathotype. AM-toxin I induced veinal necrosis on leaves of Le Lectier and General Leclerc cultivars, both susceptible to the European pear pathogen, at 5?×?10^?7 M and 10^?6 M respectively, but did not affect leaves of resistant cultivars at 10^?4 M. PCR analysis with primers that specifically amplify the AM-toxin synthetase gene detected the product of expected size in the pathogen. These results indicate that A. alternata causing black spot of European pear is identical to that causing Alternaria blotch of apple. This is the first report of European pear disease caused by the A. alternata apple pathotype. This study provides a multiplex PCR protocol, which could serve as a useful tool, for the epidemiological survey of these two diseases in European pear and apple orchards.