Phenology of infection on apple fruit by sooty blotch and flyspeck species in Iowa apple orchards and phylogenetic analysis to assess the evolutionary origins of sooty blotch and flyspeck on apple

The sooty blotch and flyspeck (SBFS) complex is comprised of more than 80 species of fungi which colonize the surface of apple fruit. The dark blotches caused by SBFS species result in economically significant damage to apple in humid production regions worldwide. Despite rapid progress in clarifying the taxonomy and ecology of SBFS fungi, there is no information available about species-specific patterns in the timing of fruit infection. A motivation for obtaining this knowledge is that it may set the stage for development of more efficient SBFS management practices, since the species that are prevalent in apparent orchards vary among geographic regions. The first objective of this study was therefore to determine whether there are species-specific patterns in the timing of SBFS infection on apple fruit. To answer this question, an experiment was conducted in six commercial apple orchards in central Iowa in 2009 and 2010. Beginning 10-21 days after petal fall, apples were covered individually by fruit bags. A subsample of apples (cv. Golden Delicious) was exposed for each of seven consecutive 2-week-long exposure periods, and then rebagged from the end of the exposure period until harvest. All individual colonies that were visible at harvest were identified using a PCR-RFLP protocol. A total of 15 species were identified. The results provided the first evidence that some SBFS species differ significantly from others in the timing of fruit infection, and found that seven SBFS species displayed the same general temporal pattern: in each species, fruit infections that resulted in formation of visible colonies at harvest peaked during the first half of the fruit development period and generally decreased thereafter. The second objective of my PhD research concerned a previously unexplored topic: the evolutionary origin of SBFS fungi. Although about 90% of SBFS species are in the order Capnodiales, evolutionary origins of major SBFS lineages in this order remain unknown. Previous phylogenetic analysis suggested that most SBFS fungi share a close evolutionary history with plant-parasitic species. The objective of this study was to assess the evolutionary origins of major SBFS lineages on apple by using ancestral state reconstruction of the 28S nuclear large subunit (LSU) region of rDNA and the second largest subunits RNA polymerase II (RPB2) region. The datasets were broadly sampled and included well-described non-SBFS as well as SBFS species in the Capnodiales. The resulting phylogeny, using a Bayesian approach, showed strong support for an evolutionary relationship between SBFS fungi and plant-parasitic fungi for some families in the Capnodiales. We performed ancestral state reconstruction with BayesMultiState, which provided strong evidence that the ancestor of Capnodiales was a plant parasitic lifestyle. Knowledge gained from this study may help to better understand the ecology and evolution of fungi that inhabit plant surfaces

First report of Pythium coloratum causing root rot on hydroponically grown lettuce (Lactuca sativa L.) in Malaysia

In April 2019, green coral lettuce (Lactuca sativa L.) showing root rot symptoms (approximately 20% disease incidence) were observed in a hydroponic commercial greenhouse located at Genting Highland, Pahang, Malaysia. Symptoms appeared as necrotic lesions on the tap and lateral roots, and affected lettuce appeared stunted and wilted. Symptomatic roots were surface-sterilized by 70% ethanol, rinsed with sterile distilled water, cut into 0.5 cm pieces, and plated on PARP-V8 selective medium (Moorman et al. 2002). The colonies produced flamentous sporangia, 20 μm yellowish and subglobose oogonia, and antheridia developed on branched stalks. Six isolates with similar morphological characteristics to Pythium coloratum (Van der Plaats-Niterink 1981) were obtained from tissue samples. The primer pair OomCoxI‐Levup/OomCoxI‐Levlo for cytochrome oxidase subunit 1 (COI) (Robideau et al. 2011) and ITS5/ITS4 for rDNA (White et al. 1990) were used for sequencing three representative isolates (PA, PB, and PC). The resulting sequences were deposited in the GenBank (COI Accession No. MW316679, MT996498, MW316680; ITS Accession No. MT659403, MT659403, MW316680). BLAST analysis of COI and ITS sequences showed 100% identity to P. coloratum strain BR621 (HQ708550 and HQ643504, respectively), and P. coloratum voucher BR621 (HQ708550.1 and HQ643504.1, respectively). Isolates PA, PB and PC grouped in a strongly supported clade (100% bootstrap) with reference strains of P. coloratum. Pathogenicity test were conducted on ten 4-week-old green coral lettuce seedlings grown hydroponically at 23 °C, 95% relative humidity with a photoperiod of 12-h. The roots were inoculated by a suspension (106 zoospores/ml) of the PB strain. Control roots were placed in sterile deionised water. After 14 days, similar symptoms to those observed in the greenhouse appeared on inoculated roots, while control plants were asymptomatic. The pathogen was reisolated from symptomatic roots and morphologically and molecularly identifed as P. coloratum. The reference isolate PB was deposited in the Microbial Collection Unit, Institute of Biosciences, University Putra Malaysia, with accession number UPMC 1451. To our knowledge, this is the frst report of P. coloratum causing root rot of lettuce in Malaysia

Enhanced nutritional programme: an innovative approach to controlling plant diseases in the tropics

Mineral nutrients are essential for the normal growth and development of both plants and microorganisms and play a crucial role in plant–pathogen interactions, which could be beneficial or detrimental to plant health. Excessive nutrient uptake causes toxicity in plants, so meeting optimal nutrient requirements is very important in the prevention of plant nutrient-deficiency symptoms. In most cases, mineral nutrients represent the primary lines of plant defense against plant pathogens and are directly affected by the plant host, pathogens, and environmental factors. In general, mineral nutrients may affect disease resistance through induced defenses, including by the production of toxins, metabolites, and lignin. The application of an enhanced nutritional programme (ENP) to minimize the deleterious effects of phytopathogens has become a hot topic of discussion around the world. Furthermore, various reports from several countries have shown that the application of mineral nutrients such as calcium (Ca), copper (Cu), and zinc (Zn) in a disease-control programme could enhance crop production and quality, suppress various plant diseases, and promote plant growth. Thus, the aim of this chapter is to discuss conventional disease control methods in the tropics with special reference to major diseases in rice, oil palm, and citrus, as well as limitations on their control. Moreover, the potential application of an ENP as part of a disease control programme in plantation crops in Malaysia is also discussed

Qualitative analysis of some bioactive components of methanolic leaf extract of M. citrifolia (Noni)

Medicinal plants offer endless opportunities for new drugs discovery due to their supremacy for the possession of phytochemicals compounds known for diverse antimicrobial activities. The world ever increasing demand for therapeutic drugs from natural products with particular interest in edible plants for safety purposes is now catching researchers’ attention. This study therefore aimed at determining the presence of some bioactive phytochemical components of methanolic leaf extract of M. citrifolia L. Qualitative screening of leaf extract has confirmed the existence of Tannins, steroids, saponins, flavonoids and alkaloids in the mixture. And these bioactive compounds correspond to phytochemicals with antimicrobial, nematicide, pesticidal, antioxidant, ant-inflammatory, cytotoxic, anti-allergy, and anti-carcinogenic properties (bioactive compounds) earlier documented by previous researchers

Molecular characterization and pathogenicity of stagonosporopsis cucurbitacearum causing gummy stem blight disease of watermelon (Citrullus lanatus L.) in Malaysia

Watermelon (Citrullus lanatus L.) is a popular fruit crop with high economic value and widely grown in Malaysia. In January 2020, gummy stem blight (GSB) has become a threat for production of watermelon in Malaysia particularly in warm and humid climates, but the causative agent of GSB infecting watermelon is unknown. This disease decreases the fresh fruit marketability. Watermelon plants cultivar Red Rocky showing varied degree (40-90%) of suspected GSB, were collected from two main watermelon growing areas in Malaysia. Initial symptoms appeared as marginal brown necrotic lesions on leaves, while on the stems showed water-soaked, necrotic lesions and exuded reddish-brown gummy exudate on the stems. A total of ten isolates were isolated from lesions on leaves and stems of watermelon plants affected by GSB. All ten fungal isolates were identified as Stagonosporopsis cucurbitacearum on the basis of morphological characteristics and phylogenetic analysis of combined sequences of the internal transcribed spacer (ITS) and β-tubulin regions. All isolates were proven to be pathogenic when inoculated on the leaves and stems of the watermelon plant and the the fungal isolates were consistently reisolated from the diseased watermelon plants confirming Koch’s postulates. Pathogenicity tests indicated that there were significant differences in virulence among the S. cucurbitacearum when inoculated on the leaves and stems of the watermelon plant. Understanding the etiology of the pathogen will help in disease management of gummy stem blight disease in Malaysia

Effect of aqueous neem leaf extracts in controlling Fusarium Wilt, soil physicochemical properties and growth performance of banana (Musa spp.)

Neem leaf extracts (NLEs) have frequently been used to inhibit plant diseases and for the development of bio-fertilizer, leading to the commercial exploitation of this tree. However, previous studies have indicated contradictory outcomes when NLE was used as an antifungal disease treatment and bio-fertilizer applied through the soil on several crops, including banana. Therefore, the present investigation was undertaken to examine the physicochemical properties of soil, the growth performance of crops, and the severity of diseases caused by Fusarium oxysporum (Foc) on Cavendish bananas treated with aqueous NLE. Banana plants associated with the fungus were significantly affected by high disease severity and symptoms index (external leaves and internal rhizome), a high infection percentage of Fusarium wilt (%), dropping off of leaves as well as rotting of the root. Meanwhile, it was observed that the application of extract significantly improved the crop height, stem diameter, root size and distribution (root surface area, root diameter, and root volume), root–shoot ratio, as well as the soil physicochemical properties (CEC, N, p, K, Ca, and Mg), which enhanced resistance to Fusarium wilt diseases. We conclude that the application of NLE solution promotes better growth of Cavendish banana plants, soil physicochemical properties, and resistance to Fusarium wilt infection

Marine-derived fungi: a promising source of halo tolerant biological control agents against plant pathogenic fungi

In this study, twenty marine-derived fungi were evaluated for their antagonistic activities against 10 economically important plant pathogenic fungi and investigated for their halo tolerance on potato dextrose agar (PDA) amended with 1%-25% NaCl. The results of dual culture tests showed that the marine Trichoderma species, T. asperellum and T. harzianum exhibited higher antagonistic effects against all plant pathogens than the other tested fungi, causing percentages of mycelial growth inhibition ranging from 59.31-100%. The results of dilution plate assays revealed that crude extracts of marine-derived fungi in the genera Emericella, Myrothecium, Neocosmospora, Penicillium and Talaromyces displayed great antifungal activity against plant pathogenic fungi at a low concentration of 1 g/L. However, the crude extract of Myrothecium verrucaria showed the best antifungal activity: more than 52% inhibition of five of the tested species of plant pathogenic fungi and complete mycelial growth inhibition of Bipolaris oryzae and Lasiodiplodia theobromae at 1 g/L. All of the tested marine-derived fungi were tolerant to NaCl at concentrations up to 7%. These results revealed marine-derived fungi possess exploitable antagonistic activities against plant pathogenic fungi through antibiosis, competition for nutrients and space and halo tolerance. Moreover, the results from this study showed their potential as novel BCAs for supporting crop production under climatic changes in the future

Energy potential of oil palm Empty Fruit Bunch (EFB) fiber from subsequent cultivation of Volvariella volvacea (Bull.) singer

EFB and EFB-based mushroom compost (SMC) from Volvariella volvacea cultivation is a promising energy feedstock because it has adequate nutrient quality. The biochemical methane potential (BMP) and calorific value (CV) of this biomass are investigated. Other analyses such as proximate, compositional, and final analysis; thermogravimetric analysis (TGA); and Fourier transform infrared spectroscopy (FTIR) are also performed. The biomass samples consist of two types of EFB, namely fibers (F) and pellets (P) and SMC from the subsequent cultivation of Volvariella volvacea, with samples FS and PS from the first cultivation and FS2 and PS2 from the second cultivation. P produces the highest biological efficiency (BE) of 28% compared to 9.83% for F. Subsequent cultivation with FS and PS then produces only 2.9 and 6.83% of BE. A higher amount of methane is measured in samples P and PS2, while better biodegradability is observed in PS2 and FS2, suggesting that subsequent cultivation is a good pretreatment of the substrate for anaerobic digestion (AD). CV is highest in F (20.57 MJ/kg), followed by P (19.06 MJ/kg), which is comparable to commercial wood pellet. Samples F, FS, and FS2 have higher ash content, which is due to higher mineral content. The cellulose composition is reduced to almost 50% during cultivation due to fungal metabolism, which is also evidenced by FTIR analysis. TGA analysis revealed that EFB-based SMC exhibits higher weight loss during combustion compared to EFB, which reduces its thermal properties. SMC of EFB is a high potential biomethane feedstock, but not recommended as a fuel pellet

First Report of Colletotrichum siamense causing Anthracnose on White Frangipani (Plumeria alba L.) in Malaysia

Plumeria alba L. is a flowering plant in the family Apocynaceae and widely cultivated in Malaysia as a cosmopolitan ornamental plant. In January 2020, anthracnose lesions were observed on leaves of Plumeria alba planted in Agricultural Farm, Universiti Putra Malaysia, in Selangor state, Malaysia. The disease mainly affected the leaves with symptoms occurring with approximately a 60% disease incidence. Ten symptomatic leaves were sampled from 3 different trees in the farm. Symptoms initiated as small circular necrotic spots that rapidly enlarged into black lesions with pale brown borders. Diseased tissues (5×5 mm) were surface-sterilized with 70% ethanol for 1 min, rinsed three times with sterile distilled water, dried on sterile filter papers, plated on PDA and, incubated at 25 °C with a 12-h photoperiod. A total of seven single-spore isolates with similar colony morphologies were obtained from tissue samples. After 7 days, the colonies raised the entire margin and showed white-to-gray aerial mycelium, orange conidial masses in the center and appeared dark brown at the center of the reverse view. The conidia were 1-celled, hyaline, smooth-walled, cylindrical with narrowing at the center, averaged (13-15 μm × 3 - 4 μm) (n=40) in size. Morphological characteristics of the isolates were similar to those detailed in taxonomic description of Colletotrichum sp. (Prihastuti et al. 2009). For molecular identification, genomic DNA of two representative isolates, PL3 and PL4 was extracted from fresh mycelium using DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) region, actin (ACT) and calmodulin (CAL) genes were amplified using ITS5/ITS4 (White et al. 1990), ACT-512F/783R (Carbone and Kohn 1999) and CL1C/CL2C primer sets (Weir et al. 2012). A BLAST nucleotide search of GenBank using ITS sequences showed 100% identity to Colletotrichum siamense ex-type culture ICMP 18578 (GenBank accession no. JX010171). ACT and CAL sequences showed 100% identity with C. siamense ex-type isolate BPD-I2 (GenBank accession no. FJ907423 and FJ917505). The sequences were deposited in GenBank (ITS: accession nos. MW335128, MT912574), ACT: accession nos. MW341257, MW341256, CAL: accession nos. MW341255 and MT919260). Based on these morphological and molecular characteristics, the fungus was identified as C. siamense. Pathogenicity of PL3 and PL4 isolates was verified using four healthy detached leaves of Plumeria alba. The leaves were surface-sterilized using 70% ethanol and rinsed twice with sterile water before inoculation. The leaves (three inoculation sites/leaf) were wounded by puncturing with a sterile needle through the leaf cuticle and inoculated in the wound site with 10-μl of conidial suspension (1×106 conidia/ml) from 7-days-old culture on PDA. Four leaves were used as a control and were inoculated only with 10-μl of sterile distilled water. Inoculated leaves were kept in humid chambers for 2 weeks at 25 °C with 98% relative humidity on a 12-h fluorescent light/dark period. The experiment was repeated three times. Anthracnose symptoms were observed on all inoculated leaves after 3 days, whereas controls showed no symptoms. Fungal isolates from the diseased leaves showed the same morphological characteristics as isolates PL3 and PL4, confirming Koch's postulates. C. siamense has been reported causing anthracnose on rose (Rosa chinensis) in China (Feng et al. 2019), Coffea arabica in Thailand (Prihastuti et al. 2009) and mango leaf anthracnose in Vietnam (Li et al. 2020). To our knowledge, this is the first report of Colletrotrichum siamense causing leaf anthracnose on Plumeria alba in Malaysia. Accurate identification of this pathogen provides a foundation in controlling anthracnose disease on Plumeria alba

Phytopathogenic organisms and mycotoxigenic fungi: Why do we control one and neglect the other? A biological control perspective in Malaysia

In this review, we present the current information on development and applications of biological control against phytopathogenic organisms as well as mycotoxigenic fungi in Malaysia as part of the integrated pest management (IPM) programs in a collective effort to achieve food security. Although the biological control of phytopathogenic organisms of economically important crops is well established and widely practiced in Malaysia with considerable success, the same cannot be said for mycotoxigenic fungi. This is surprising because the year round hot and humid Malaysian tropical climate is very conducive for the colonization of mycotoxigenic fungi and the potential contamination with mycotoxins. This suggests that less focus has been made on the control of mycotoxigenic species in the genera Aspergillus, Fusarium, and Penicillium in Malaysia, despite the food security and health implications of exposure to the mycotoxins produced by these species. At present, there is limited research in Malaysia related to biological control of the key mycotoxins, especially aflatoxins, Fusarium‐related mycotoxins, and ochratoxin A, in key food and feed chains. The expected threats of climate change, its impacts on both plant physiology and the proliferation of mycotoxigenic fungi, and the contamination of food and feed commodities with mycotoxins, including the discovery of masked mycotoxins, will pose significant new global challenges that will impact on mycotoxin management strategies in food and feed crops worldwide. Future research, especially in Malaysia, should urgently focus on these challenges to develop IPM strategies that include biological control for minimizing mycotoxins in economically important food and feed chains for the benefit of ensuring food safety and food security under climate change scenario