Fusarium mangiferae localization in planta during initiation and development of mango malformation disease

Mango malformation disease (MMD), caused by Fusarium mangiferae, is a major constraint to mango production, causing significant yield reduction resulting in severe economic impact. The present study characterizes fungal localization in planta during initiation and development of vegetative and floral malformation. Young mango trees were artificially inoculated with a green fluorescent protein (GFP)‐expressing strain of F. mangiferae. Shoots and buds were sampled periodically over a period of more than a year and localization of the GFP‐expressing fungi was determined using confocal microscopy. Fungal localization appears to be epiphytic: mycelia remained in close contact with the plant surface but did not penetrate the tissue. In vegetative malformation and in young inflorescences, the fungus was confined to protected regions between scales, young leaf bases and buds. Fungal colonization was only very rarely detected on open leaves or on exposed shoot sections. In developed flowers, mycelia were localized mainly to protected regions at the base of the flower organs. Upon development of the inner flower organs, specific mycelial growth occurred around the anthers and the style. Mycelial penetration through the stylar tract into aborting carpels was observed. For several months, mycelia were confined to the surface of the organs and were not detected within plant tissues. Only at later stages, transient saprophytic growth of the fungus was detected causing the malformed inflorescences to senesce and collapse, concurrent with dispersion of conidia. Implications of the present study on MMD in natural field infections are discussed.     

Antifungal activity of Datura stramonium, Calotropis gigantea and Azadirachta indica against Fusarium mangiferae and floral malformation in mango

Floral malformation caused by Fusarium mangiferae is a serious threat to mango cultivation in various countries. Different long-term measures suggested to control it were found to be unsuccessful. Present studies clearly showed strong antifungal activity of a concoction brewed from Datura stramonium, Calotropis gigantea, Azadirachta indica (neem) and cow manure (T₁) followed by methanol-water (70/30 v/v) extracts of Datura stramonium, Calotropis gigantea and Azadirachta indica (T₂) against Fusarium mangiferae. Optimal control of floral malformation was found in trees sprayed with T₁ followed by T₂ at bud break stage and again at fruit set stage when compared with the control. All the malformed buds or panicles completely dried two days after foliar spray with T₁ or T₂. In the trees treated with T₁ at fruit set stage, flower abscission was observed from the fourth day after spraying and all flowers dropped by the ninth day without requiring any manual de-blossoming, whereas in the control, the malformed panicles remained green and competed with the growing fruits for plant nutrients. In vitro culture of fresh malformed tissues in MS media along with T₁ or T₂ showed no growth of any fungus in the media. However, in vitro culture of the completely dry malformed tissues in MS media after foliar treatment with T₁ or T₂ revealed growth of F. mangiferae on the twenty fifth day indicating that the concoction-brewed compost (T₁) or methanol-water (70/30 v/v) extracts (T₂) could not completely eliminate the pathogen but helped in controlling malformation by suppressing the activity of F. mangiferae. Mango trees sprayed with T₁ and T₂ revealed significant differences in percent fruit set and retention when compared with the control. This could be due to observed higher levels of nitrogen, phosphorus, potassium, calcium, magnesium, copper, zinc, iron and manganese in T_1, followed by T₂ when compared with T₃ (control). Among the different fruit quality parameters analysed, the total flavonoids were found to be significantly higher in T₁ and T₂ when compared with T_3. The study proved that the concoction-brewed compost (T₁) is effective, inexpensive, easy to prepare and constitutes a sustainable and eco-friendly approach to control floral malformation in mango when it is sprayed at bud break stage and again at fruit set stage. In this present study, exogenous treatment of emerging buds with (T_c) further proved that with increase in the number of malformed panicles/tree the number of buds developing into healthy panicles/tree decrease.     

Epidemiological aspects of mango malformation disease caused by Fusarium mangiferae and source of infection in seedlings cultivated in orchards in Egypt

Mango malformation, caused by the fungus Fusarium mangiferae , is one of the major diseases of this crop occurring worldwide. This study was conducted to investigate aspects of the epidemiology, survival and spread of the pathogen in general and specifically in seedlings, the majority of which are cultivated in infected orchards in Egypt. Survival of conidia of a representative isolate (506/2) declined very rapidly in soil under summer conditions (1·6 weeks for 50% population decline), but significantly less in controlled and winter conditions (17·9 and 15·0 weeks, respectively, for 50% population decline). Likewise, inoculum survival in naturally infected panicles on the soil surface declined faster than in those buried at 30-cm depths. Natural infections were evaluated on fruits and seeds in a heavily infected and a healthy orchard. In infected trees, the skins of all sampled fruits within a 2-m radius of infected panicles were infected, but the pathogen was not detected in the seeds, seed coats or flesh. The pathogen was not detected in any parts of fruits from a healthy orchard. Vegetatively malformed mango seedlings, growing under infected trees bearing infected panicles, were sampled in two locations in Egypt to determine whether infection in seedlings was systemic (evenly distributed within plant tissue) or whether the pathogen originated from malformed panicles. According to PCR-specific primer amplification, the pathogen was detected in 97% of seedling apical meristems, declining gradually to 5% colonization in roots. It was concluded that inoculum of the pathogen originates from infected panicles and affects seedlings from the meristem, with infections descending to lower stem sections and roots. Minor infections of roots may occur from inoculum originating from infected panicles, but the pathogen is not seedborne.     

Colonization of the vegetative stage of rice plants by the false smut fungus Villosiclava virens,as revealed by a combination of species‐specific detection methods

Rice false smut disease caused by the ascomycete fungus Villosiclava virens (Clavicipitaceae) reduces rice yield worldwide. It invades rice panicles and forms dark‐green false smut balls composed of thick‐walled conidia. Although the infection process during the booting stage is well studied, its infection route before this is unclear. It was hypothesized that the thick‐walled conidia in soil penetrate rice roots, and the fungus latently colonizes roots and tiller buds at the vegetative stage. This hypothesis was tested using species‐specific detection methods. First, real‐time PCR with species‐specific primers and probe was used to estimate thick‐walled conidial number in the paddy field soil. Secondly, nested PCR with species‐specific primers showed that fungal DNA was detected in roots and shoot apices of rice plants in the vegetative stage. Thirdly, colourimetric in situ hybridization with a species‐specific oligonucleotide probe targeting 18S rRNA suggested that sparse mycelia or tightly condensed mycelia were present on the external surface of tiller buds enveloped by juvenile leaf sheaths at the vegetative stage. Thin hyphae were found around leaf axils at the surface of elongated stems at the heading stage, and the fungal hyphae grew in the rice root tissues. In addition, it was demonstrated that eGFP‐tagged transformants of the fungus invaded rice roots and colonized the surface of roots and leaf sheaths under artificial conditions.     

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium mangiferae associated with mango malformation

Fusarium mangiferae is a major causal agent of mango malformation disease (MMD) worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for quantitative detection of F. mangiferae in China. The LAMP primer set was designed based on a RAPD marker sequence and positive products were amplified only from F. mangiferae isolates, but not from any other species tested, showing a high specificity of the primer sets. The detection limit was approximately 2.26 × 10⁻⁴ ng/μl plasmid DNA when mixed with extracted mango DNA. Quantification of the pathogen DNA of MMD in naturally collected samples was no significant difference compared to classic real-time PCR Additionally, RealAmp assay was visual with an improved closed-tube visual detection system making the assay more convenient in diagnostics.     

Mango Eriophyid mites in relation to inflorescence

Following the appearance of inflorescence malformations on mango trees in some orchards in Israel, a survey was carried out of the presence of Eriophyid mites on mango, and their possible effect on inflorescence malformation was studied. Two Eriophyid species were recorded for the first time from this area. One of them,Erio-phyes mangiferae, associated with mango trees, has no direct effect on inflorescence malformation, but its interaction with another biotic factor is not excluded.     

<Emphasis Type="Italic">Fusarium mangiferae</Emphasis> associated with mango malformation in the Sultanate of Oman

Mango malformation, caused by Fusarium mangiferae, represents the most important floral disease of mango. The first symptoms of this disease were noticed in the beginning of 2005 in plantations at Sohar in the Sultanate of Oman. The affected inflorescences were abnormally enlarged and branched with heavy and dried-out panicles. Based on morphology and DNA-sequence data for the genes encoding translation elongation factor 1α and β-tubulin, the pathogen associated with these symptoms was identified as F. mangiferae.     

Use of GUS Transformants of Fusarium subglutinans for Determining Etiology of Mango Malformation Disease

ABSTRACT Fusarium subglutinans has been associated with mango floral and vegetative malformation, although confusion exists regarding the etiology of the disease. A wild-type isolate of F. subglutinans causing mango malformation disease was transformed with the GUS (beta-glucuronidase) reporter and hygromycin resistance genes. Five stable transformants were isolated containing varying copy numbers at different integration sites. Specific GUS activity was quantified for the transformants, whereas no activity was recorded for the wild-type isolate. The transformants and the wild-type isolate were inoculated into healthy mango floral and vegetative buds. Typical symptoms of misshapen shoots with short internodes, stubby leaves, and bunchy, malformed inflorescences were observed 6 to 8 weeks following inoculation. The presence of GUS-stained mycelium of the pathogen viewed microscopically within infected plant organs provided unequivocal evidence that F. subglutinans is indeed a causal agent of mango malformation disease.     

Effects of panicle development stage and temperature on rice panicle blast infection by Magnaporthe oryzae and visualization of its infection process

Panicle blast, caused by the fungus Magnaporthe oryzae (syn. Pyricularia oryzae), directly contributes to yield loss in the field. The effects of panicle development stage and temperature on panicle blast were studied and the infection process of M. oryzae in panicles was visualized. Rice panicles at different development stages from three rice cultivars were inoculated with a conidial suspension in vitro. The rice cultivar Lijiangxintuanheigu was highly susceptible to panicle blast at 5 days postinoculation (dpi) when the pulvinus distance was 15–20 cm. Nanjing 9108 was moderately susceptible to panicle blast when the pulvinus distance was 8–10 cm, but Yliangyou 800 was resistant. The effect of temperature on panicle blast was determined under 22–35 °C temperature treatments. Inoculated panicles placed at temperatures of 28 and 30 °C showed the highest lesion grade based on lesion length at 5 dpi. The infection process of M. oryzae in rice panicles was observed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). M. oryzae initially formed the appressorium to invade through the epidermis of rice panicles at 24 hours postinoculation (hpi). As the disease progressed, the invasive hyphae formed dense mycelial networks in the inner parenchyma cells at 60 hpi. Our results will contribute to the understanding of panicle development stage and temperature effects on panicle blast and improve resistance evaluation methods. Additionally, visualization of the infection process by CLSM and TEM are valuable methods to observe M. oryzae invasive hyphae inside rice panicle cells.     

First report of sclerotinia rot of mango caused by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in Japan

A panicle blight with sclerotia was found on mango (Mangifera indica L.) in Okinawa Prefecture, Japan, in March 2016. Water-soaked lesions with white mycelia developed on panicles in the flowering stage; softening and decay of panicles was followed by formation of sclerotia. The fungus isolated from these sclerotia was identified as Sclerotinia sclerotiorum based on morphology and analysis of rDNA-ITS sequences. The isolate reproduced the symptoms on mango panicles in an inoculation test and was reisolated from flower stalks. This is the first report of sclerotinia rot (kinkaku-byo in Japanese) on mango caused by S. sclerotiorum in Japan.     

Molecular diagnosis of mango malformation disease and phylogeny of Fusarium mangiferae

Mango malformation disease (MMD) caused by various Fusaria, including the fungus Fusarium mangiferae, is difficult to diagnose and cannot be controlled effectively. In this study a PCR diagnostic tool was developed for detection of F. mangiferae by generating primers which flank fungus-specific sequences. The assay is suitable for both in-vitro and in-vivo tests, and is capable of detecting 10?pg of the fungal DNA. Relatively high genetic variation was discovered between various isolates of the pathogen based on amplified fragment length polymorphism (AFLP) analyses. In addition, some isolates of F. mangiferae (including the accession numbers FMS-123 and NRRL 25226), were found to be quite distinct from most F. mangiferae accessions. Likewise, representative MMD-associated Fusaria from the Asian and American clades of the Gibberella fujikuroi species complex were further differentiated by AFLP analyses.     

Histopathological aspects of mango resistance to the infection process of Ceratocystis fimbriata

Mango wilt, caused by Ceratocystis fimbriata, is one of the most important diseases affecting mango yields in Brazil. Information regarding the infection process of C. fimbriata in the stem tissues of mango from different cultivars and the basis of host resistance to the pathogen is rare in the literature. Thus, the objective of the study was to investigate how infection by two isolates of C. fimbriata can be affected by mango cultivar‐specific mechanisms of resistance. Disease progress on the inoculated stem tissues of the mango cultivars was evaluated and stem sections were obtained from the site of inoculation and prepared for histopathological observations using light microscopy. The factors mango cultivars and C. fimbriata isolates and their interaction were significant for all measures of disease development. Plants from the cultivars Espada, Haden and Palmer inoculated with isolates of C. fimbriata were more susceptible, whereas plants from the cultivars Tommy and Ubá were moderately resistant and resistant, respectively. Histopathologically, fungal isolates apparently massively colonized the stem tissues of plants from the susceptible cultivars Espada, Haden and Palmer, starting from the collenchyma and moving in the direction of the cortical parenchyma, xylem vessels and pith parenchyma. By contrast, on stem tissues of plants from the resistant cultivars Tommy Atkins and Ubá, most of the cells reacted to C. fimbriata infection by accumulating amorphous material. The results from the present study strongly indicated the importance of phenolic compounds for mango cultivar resistance against infection by Brazilian C. fimbriata isolates.     

A nested multiplex PCR for species-specific identification and detection of Botryosphaeriaceae species on mango

Lasiodiplodia theobromae (Pat.) Griff. & Maubl, Neofusicoccum parvum Pennycook & Samuels, N. mangiferae Syd. & P. Syd., and Fusicoccum aesculi Corda, all anamorphs of Botryosphaeriaceae species, are the causal agents of mango stem-end rot and fruit rot in Taiwan. Identification of these fungal species based on morphology has not been easy due to their extensive plasticity for some of the morphological characters. To aid reliable identification of Botryosphaeriaceae species associated with mango fruits, four pairs of species-specific primers were designed according to sequences of the ribosomal internal transcribed spacers (ITS), and a rapid method was established based on nested multiplex polymerase chain reaction (PCR) in this study. To perform the analysis, PCR was first run with ITS1 and ITS4 as the primers, followed by a second PCR with the addition of all four sets of species-specific primers. With this method, a low limit of 100?fg^-1?pg of purified fungal DNA was detectable. It could also successfully detect L. theobromae, N. parvum, N. mangiferae and F. aesculi in total DNA extracted from inoculated mango fruits. This assay provides a rapid and sensitive method for the identification of Botryosphaeriaceae species and diagnosis of mango fruit rot and stem-end rot as well.     

Integration of cultural and mechanical practices for management of the mango mealybug <Emphasis Type="Italic">Drosicha mangiferae</Emphasis>

Mango (Mangifera indica L.) is one of the most economically important trees which has been cultivated for several centuries. The tree is susceptible to insect damage and is attacked by leaf eaters, termites, root grubs, sapsuckers, gall formers, stem borers, pod borers and fruit borers. The mango mealybug Drosicha mangiferae Green (Hemiptera: Monophlebidae) is one of the most destructive pests in the Indo-Pak subcontinent, sometimes causing premature fruit drop. A combination of several management strategies such as use of entomopathogens, chemical insecticides and burning of adult females has been used to control D. mangiferae. Overreliance on insecticides has eliminated natural enemies, and we were therefore interested in investigating the impact of alternative insect pest management strategies such as cultural and mechanical techniques on D. mangiferae. The results of our studies suggested that different bands significantly stopped the upward movement of overwintering and surviving populations of D. mangiferae. To augment the bands’ results, mounding the tree with debris on a plastic sheet once a year was the most effective technique and resulted in maximum reduction of the nymphal population of D. mangiferae. The newly developed Haider band—designed in our laboratory in the present studies—was very successful in the field in controlling the upward movement of newly hatched nymphs on trees: only 0.8% of the nymphs crossed the band. Only 7% to 10% of the nymphs crossed plastic sheets, polyethylene sheets and funnel bands. The cotton and gunny bag (jute) bands were the least effective: 42% of the nymphs managed to cross them. The results suggested that employing cultural practices and bands together could reduce the need for insecticide treatments and offer a sustainable method for D. mangiferae control.     

Impact of carrot resistance on development of the <Emphasis Type="Italic">Alternaria</Emphasis> leaf blight pathogen (<Emphasis Type="Italic">Alternaria dauci</Emphasis>)

The interaction between Alternaria dauci and two carrot cultivars differing in their resistance to leaf blight was investigated by microscopy. The fungal development between 1 and 15 days post-inoculation was quite similar in the susceptible cv. Presto and the partially resistant cv. Texto: After conidial germination, leaf adhesion of the pathogen was achieved with mucilaginous filaments; hyphae penetrated the leaves directly with/without the formation of appressoria-like structures or via stomata; the fungus spread by epiphytic hyphae with hyphopodia and subcuticular mycelia. Intense necrotic plant cell reactions occurred under the fungal structures. At 21 days post-inoculation, typical features of fungal development were noted for each cultivar: growing hyphae emerged from stomata in cv. Presto, whereas conidiophores without conidia were observed in cv. Texto. Leaf tissues of both cultivars were strongly damaged and vesicle-like structures (assumed to be plant phenolics) were abundantly present between mesophyll cells. A real-time PCR method was developed for in planta quantification of A. dauci. Between 1 and 15 days post-inoculation, the fungal biomass was equivalent in the two cultivars and was about fourfold higher in cv. Presto than cv. Texto at 21 and 25 days post-inoculation. Taken together, our results indicated that A. dauci was able to colonize both cultivars in a similar manner during the first steps of the interaction, then fungal development in the partially resistant cultivar was restricted due to putative plant defence reactions. The results of this study enhance the overall understanding of infection processes in the A. dauci-carrot pathosystem.     

Ceratocystis fimbriata‐induced changes in the antioxidative system of mango cultivars

Mango wilt is one of the most important diseases affecting mango production, yet the physiological mechanisms underlying host responses to this fungal infection are poorly known. The disease attenuation displayed by some mango cultivars might be related to higher antioxidant capacities in leaves. To test this hypothesis, the activities of a range of enzymes (e.g. superoxide dismutase and catalase) and metabolites (e.g. ascorbate and glutathione) involved in the antioxidant system, as well as some markers for oxidative stress (e.g. hydrogen peroxide and malondialdehyde equivalents), were evaluated for 30 days after the inoculation of mango plants with Ceratocystis fimbriata. Tommy Atkins plants displayed higher values for the upward, downward and radial fungal colonization of the stem tissues than Ubá plants. In contrast to the working hypothesis, Tommy Atkins plants exhibited more prominent increases in enzyme activities and metabolite concentrations related to oxidative stress responses. These increases were minimal, if any, in Ubá plants. These results suggest that Tommy Atkins plants are less effective at restraining fungal spread through vascular tissues and thus trigger leaf antioxidant defences to cope with the developing stress.     

Susceptibility of grapevine buds to infection by powdery mildew Erysiphe necator

Bud colonization and perennation of powdery mildew ( Erysiphe necator ) was studied by inoculating shoots of grapevine ( Vitis vinifera cv. Carignane) at different phenological stages. Disease incidence and severity assessments indicated that buds were most susceptible at the three- to six-unfolded-leaf stage. Incidence of powdery mildew colonies on the surface of buds collected from these shoots 7 weeks postinoculation was highest at these stages (68 and 62%, respectively), which indicates that colonization of the bud interior via the infected bud surface is likely to occur within this period. Histological analyses of buds revealed hyphae with haustoria, conidiophores and conidia on all parts of the bud interior except for the meristems. In particular, trichomes were frequently parasitized by haustoria. In total, 13·2% of all buds analysed, and 32·3% of all buds originating from shoots inoculated at the three-unfolded-leaf stage, were infected by E. necator . In the spring of the following year, buds from inoculated shoots yielded 18 flag shoots (1·6% of all emerging shoots). These primary infections caused an epidemic 28 days after the appearance of the first flag shoot. A linear regression analysis on the frequency of infections of the bud exterior, bud interior and flag shoots revealed that incidence of external bud infection in the first season is strongly correlated with flag shoot incidence in the following season ( R ² = 0·94). Hence predictions of flag shoot incidence may be reliably based on the incidence of infection on the outer bud scales in the preceding season.     

The mango sudden decline pathogen, Ceratocystis manginecans, is vectored by Hypocryphalus mangiferae (Coleoptera: Scolytinae) in Oman

In Oman, the bark beetle Hypocryphalus mangiferae is closely associated with trees affected by mango sudden decline disease caused by Ceratocystis manginecans. Although it has previously been assumed that this beetle plays a role in the dispersal of the pathogen, this has not been established experimentally. The aim of this study was to determine whether H. mangiferae vectors C. manginecans from infected to healthy mango trees. A survey conducted in northern Al Batinah region of Oman revealed that H. mangiferae was closely associated with mango sudden decline disease symptoms and it was found on trees in the early stages of the disease. Healthy, 2-year-old mango seedlings were exposed to H. mangiferae collected from diseased mango trees. Seedlings were infested by the bark beetles and after 6 weeks, typical mango sudden decline disease symptoms were observed. Ceratocystis manginecans was isolated from the wilted mango seedlings while uncolonized control seedlings remained healthy. The results show that H. mangiferae vectors C. manginecans in Oman and is, therefore, an important factor in the epidemiology of this disease.     

Bait twig method for soil detection of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>, causal agent of white root rot of Japanese pear and apple,at an early stage of tree infection

Mulberry twigs were inserted into the soil as bait to detect Rosellinia necatrix at an early stage of tree infection in the orchard. R. necatrix was frequently trapped on twigs near the trunk base at soil depths of 6–20 cm within 10–20 days in May–July, suggesting that the incubation period was dependent on soil temperature. Subsequently, we inserted twig in the soil around healthy-looking trees in naturally infested orchards. R. necatrix was trapped from 80.0% of Japanese pear and 75.0% of apple trees that later proved to be infected. This bait twig method facilitated quicker diagnosis of white root rot on Japanese pear and apple at early stages of infection and can be used to detect recurrence of the fungus after fungicide treatment.     

Susceptibility of grapevine tissues to Neofusicoccum luteum conidial infection

This study investigated the ability of Neofusicoccum luteum to infect wounded shoots, trunks, pruned cane ends, leaf surfaces, buds, berries and roots, and its further progression into stem tissues. All tissue types were susceptible to infection except roots, with highest incidences in trunks (100%), cane ends (100%), shoots (92%) and buds (88%), indicating that in New Zealand, N. luteum is primarily a trunk and shoot pathogen. In trunks, there were no external symptoms, although N. luteum could be reisolated from 60 to 70 cm acropetally from the inoculation site after 4 months, by which time the pathogen had progressed into side shoots which became necrotic. Wounded and non‐wounded buds became infected; most were killed, with basipetal progression of the pathogen into the supporting shoots. Berries wounded and inoculated at the pre‐bunch closure stage were susceptible to N. luteum infection, with isolation incidence increasing over the season and peaking at harvest, when infected berries became mummified and produced pycnidia with many conidia. The pathogen was also able to progress from berries into bunch stems and supporting canes. Results from this research have indicated that N. luteum infection can occur in all aerial grapevine tissues and progress to young stem tissues where it causes wood necrosis. Growers should remove mummified berries from vineyard trash to ensure that pruning and trimming times do not coincide with rainy periods when conidia are released and dispersed. Furthermore, the susceptibility of buds to N. luteum infection indicates the need for fungicide sprays before budburst in spring.