53 comments

  1. Don’t you let water go from one pit to the other pits with a ( PVC ) pipe. If it’s a disease, it’s also running through your ( PVC ) not only your scoop. Your connection pipe is also a large problem with disease. It will keep going with transferring water. Spores from azolla and small amounts of plants will go through your ( PVC ) there is no way to stop all bacteria from going through the pipe.

  2. Be tempting to try and pour some bleach in one of the pits that is dying and see if it clears up. You have had many problems with the pits, at what point is the savings not worth the investment?

  3. Getting painful to watch you and that Azolla experiment… Many things just do not grow well in the Philippines… Much of that stuff in your garden looked great and then just quickly croaked in a similar fashion…

  4. I hate to be negative but stick to one thing instead of trying so many different projects. Once you have one perfected and actually making you a profit then try another.

  5. I really think it’s too much sun and too hot. It’s just baking it in the sun. That corn and that little bit of shade net isn’t enough. When it was doing good after the die off wasn’t it raining a lot and overcast? If you build the tilapia fingerling grow out pits up near the old worm casting pit… Before you add any fingerlings in it, throw a few scoops in them. I’d bet they’d do good up in the shade. I doubt it’s anything to do with disease or fertilizers. It’s a shade issue in my opinion. Needs an actual roof or put the shade net back up and add more coconut leaves on the shade net to block out the majority of the sun. Not just a few leaves here and there. Overlap them the whole way along for lots of shade. Just a suggestion. Hopefully you can figure it out soon.

  6. When the pits get harvested I noticed that it’s always the ends of the pits that Azolla is taken from leaving old Azolla in the middle of the pits. Could this start a chain reaction of dying Azolla?

  7. Brian i think you have to go back to the beginning and go through your videos your first few Azolla pits were doing great if i remember correctly you didn’t have this problem until you added more Azolla pits something changed their maybe something in the soil i’m only looking at it through a mechanics eyes always go back to what you did something you could try is drain all the pits let them sit for a week in the sun that should kill it off if it’s a fungus i wouldn’t use bleach it might wreck your tarp then fill one at a time okay that’s my two cents

  8. maybe also wash the Azolla and the scoop to your doing a good job there Brian its very hard though could it be the plastic you using being contaminated ? you never rinse it you use it strait after buying it ??

  9. You need water tests and soil test on every thing. What ur doing now is blind testing. Just because you have a new well doesn’t mean the water is good, it could be highly acidic. You need proper tests on every thing, and I know it is hard to do there at the best of times. cheers….

  10. I found this research paper on the internet. It is from India. They have a brown rot there as well. It was determined by researchers that the Azolla was infected by a Fungus.
    Part 1:

    NEW RECORD OF BROWN ROT DISEASE IN WATER FERN AZOLLA

    MICROPHYLLA (AZOLLACEAE): LOSS OF IMPORTANT BIO-RESOURCE

    S. DEY, M. HORE, J. BISWAS, M. BISWAS, B.K. MANDAL, P. DAS & S. GUPTA

    Pteridology and Palaeobotany Section, Department of Botany,

    University of Kalyani, Kalyani- 741235, West Bengal, India

    e-mail id: [email protected]

    Key words: cottony white mycelium; yellow pigmentation; macroconidia;

    microconidia; chlamydospores; pathogenic fungus; Fusarium thapsinum

    ABSTRACT

    Severely infected water fern, Azolla microphylla Kaulf., has been observed in

    natural habitats in different districts in the lower Gangetic plain of West Bengal,

    India. The infected plants have turned dark brown and been rotted entirely within

    5-7 days, causing loss of natural resource as the plant species has immense

    commercial value. Specific growing colonies with cottony white mycelium,

    yellow pigmentation on the cultured medium, macro- and micro- conidial

    features, and absence of chlamydospore indicate the causal pathogen to be

    Fusarium thapsinum Klittich., Lesile, Nelson, Marases. The identification of the

    causal pathogen has been authenticated by standard pathogenicity test (Koch’s

    Postulates). Extensive literature survey reveals that this is a new record of brown

    rot disease in A. microphylla.

    INTRODUCTION

    Azolla Lam. is a free-floating, fast growing water fern. The plant is widely distributed in

    tropical, subtropical, and warm temperate zones in different countries of the World

    including India (Wagner, 1997). Here Azolla spp. are naturally available, mostly on moist

    soils, freshwater ditches, ponds, lakes, sluggish rivers and marshy lands. Azolla is treated

    as a ‘green gold mine’, because of its high nutritive value and it is considered as ‘super

    plant’ due to its fast-growing capacity (Wagner, 1997). They can even grow in nitrogen

    deficit areas and can double the biomass in 3-5 days because of its unique symbiotic

    relationship with the nitrogen fixing endophytic blue-green cyanobacterium, Anabaena

    azollae Sterberger (Wagner, 1997).

    High nutritive values (Buckingham et al., 1978; Alcantara & Querubin, 1985; Paoletti

    et al., 1987; Lejeune et al., 2000; Alalade & Lyayi, 2006) and huge productivity through

    easy cultivation methods (Dao & Tran, 1979; Lumpkin & Plucknett, 1982; Liu et al.,

    2008) are the key factors for utilizing Azolla as one of the most promising aquatic plants

    for livestock feed (Becerra et al., 1995; Alalade & Lyayi, 2006; Cagauan & Pullin, 1991;

    Shiomi & Kitoh, 2001; Fiogbe et al., 2004), and as an effective natural fertilizer to

    increase rice yield successfully (Peters, 1978; Tung & Shen, 1985; Watanabe & Liu,

    1992; Yadav et al., 2014). The plant has been employed in experiments during space and

    planetary travel (Liu et al., 2008; Carrapico, 2002; Katayama et al., 2008), and used for

    the production of non-polluting high energy biofuel (Peters et al., 1976; Newton, 1976;

    Das et al., 1994; Hall et al., 1995), especially biodiesel (Salehzadeh et al., 2014). The

    plant can act as reducer of various greenhouse gases by ammonia volatilization

    FERN GAZ. 20(6):245-254. 2017 245

    (Watanabe & Liu, 1992), by inhibiting methane production from rice field (Prasanna et

    al., 2002), and noticeably by sequestering atmospheric CO2(Brinkhuis et al., 2006). In

    addition, weed control (Krock et al., 1991), mosquito repellence (Ansari & Sharma

    ,1991), phyto-remediation from waste water (Jain et al., 1989; Saxena, 1995; Costa et

    al., 1999; Antunes et al., 2001; Khosravi et al., 2005; Umali et al., 2006; Rai, 2008; Costa

    et al., 2009; Rai & Tripathi, 2009; Elmachliy et al., 2010; Rai, 2010a, b; Sood et al.,

    2012) and soil (Umali et al., 2006; Cohen et al., 2002 and Mashkani & Ghazvini, 2009)

    are added approaches for utilizing this vital bio-resource.

    With view to the many valuable uses of Azolla, the present authors have attempted

    to explore the species under various abiotic stresses through experimental set-up by

    culture of collected specimens from natural habitats of the lower Gangetic plains of West

    Bengal, India. During field survey, it has been observed that among the available Azolla

    species, A. microphylla Kaulf. grows dominantly over others and it can resist direct

    sunlight in summer. However, the species has been found to be severely infected by a

    fungal pathogen at the end of winter season (January-March) in most of the collection

    sites, which disrupts normal growth of the plant and ultimately causes death within 5-7

    days.

    From the available literature, it seems that pathogenic reports on Azolla spp. are rather

    meagre (Sasi et al., 1982; Kannaiyan, 1985; Kannaiyan & Kumar, 2005; Lee et al., 2011;

    Banihashemi, 2014). The present communication describes a new causal organism for

    A. microphylla, which may help in identifying suitable measure(s) to protect this

    important natural resource.

    MATERIAL AND METHODS

    Fresh and infected samples of Azolla microphylla Kaulf. were collected from different

    sites of three districts, namely, North 24 Parganas (22.936 N, 88.392 E; 22.825 N, 88.786

    E), South 24 Parganas (22.021 N, 88.615 E), and Nadia (22.983 N, 88.445 E) in the lower

    Gangetic plain of West Bengal.

    The morphological studies of infected and non-infected plants were made by hand

    lens and stereo zoom microscope (model LEICA S8APO). The anatomical details of

    infected plants and that of the isolated pathogen were thoroughly studied under light

    microscope (model Leitz Laborlux S). Macro and micro photographic documentations

    were made using Nikon D3200 DSLR and EC3 scientific cameras. To identify the

    pathogen from morphological characteristics, first the isolations were made from the

    frond of the naturally infected A. microphylla plant using a sterile inoculating loop and

    plated onto 2.25% Potato Dextrose Agar (PDA). A total of five replicas of culture and

    three replicas of control plates were incubated at 25˚C temperature in complete darkness.

    After 2–3 days of inoculation, the fungal colony was observed to grow in each of the

    five replications of cultured plates but no changes were observed in control plates. The

    colony morphology, growth pattern, size and shape of macro- and micro- conidia were

    characterized thoroughly.

    To confirm the identity of the pathogen, the standard pathogenicity test (Koch’s

    Postulates) was performed stepwise. Student’s t-test was done to compare the variations

    in two sets of data of pathogens detected from inoculated

  11. Continued part 2:

    10.73±3.31×2.48±0.33 µm, 13.27±1.98 x 4.85±0.48 µm, and 7.17±0.77 x 3.37±0.18 µm

    respectively (Table 1).

    The macroconidia were found abundantly in aerial parts of the hyphae of the infected

    leaf; 3-5 septate, but rarely 6; straight to falcate, thin walled, hyaline; slightly curved and

    tapering in a banana shape (Figure 1S). The average size of macroconidia is of

    33.10±5.92 x 3.62±0.28 µm (Table 1). The apical cell of each macroconidia was conical,

    whereas the basal cell was relatively ill-developed and not the usual foot-shaped. The

    average length of the apical cell (6.47±0.77 µm) was longer than the penultimate cell

    (5.29±0.83 µm) (Table 1). It is significant to note that no chlamydospore was found in

    any one of the culture plates.

    The growth pattern of the cultured pathogen onto PDA media and the occurrence of

    distinctive banana-shaped macroconidia and specific types of microconidia indicate that

    the causal pathogen is Fusarium Link (Moretti, 2009). Fusarium is a well-known plant

    pathogenic fungus, which is soil borne in nature and causes severe plant diseases around

    the world (Agrios, 2005). The Fusarium strain that produces diffusing yellow pigment

    was separated as a new species, F. thapsinum Klittich., Lesile, Nelson, Marases

    (Teleomorph, Gibberella thapsina) (Klittich et al., 1997, Summerell et al., 2003; Leslie

    & Summerell, 2006).

    The characteristic features of cottony white mycelium, yellow pigmentation onto

    pure culture PDA medium, absence of chlamydospores, shape and size of macro- and

    microconidia corroborate with the diagnostic features of the holotype BPI 737885

    (Klittich et al., 1997) and hence identify the species epithet of the present causal pathogen

    as F. thapsinum.

    After seven days incubation in the pathogenicity tests, identical disease symptoms

    with brown-coloured lesions and protruding cottony white mycelium were observed only

    on the inoculated plants (Figure 1V) and the fungus was consistently re-isolated from

    lesions. The identity of the re-isolated pathogen was confirmed as F. thapsinum again

    by pure culturing onto PDA medium (Figure 1W) maintained at similar conditions

    mentioned before and subsequent microscopic examinations of the hyphae with

    DEY et al.: BROWN ROT DISEASE IN AZOLLA MICROPHYLLA 249

    Figure 1(A.-W.): Non-infected, and infected Azolla microphylla plant showing

    symptoms of brown rot disease, cultured colony of causal pathogen, and microscopic

    observation of different features of causal organism (bar= 0.1 mm except otherwise

    mentioned). A. & B. Naturally infected plant on pond (A) and in rice field (B); C.

    Infected plant grown in tank at experimental garden of Botany Department, Kalyani

    University; D. Non-infected plant; E. Infected root showing brown coloration; F. Brown

    coloured spots on leaf (arrow marked) at initial stage of infection; G. Gradual

    enlargement of infected area on leaf; H. Leaf showing bloch notch symptom; I. Entirely

    rotten plant at final stage of infection; J. Cottony white mycelial growth on infected leaf;

    K. Transverse section of infected leaf showing hyphal infiltration; L. Growth pattern of

    pure cultured colony showing white mycelium and diffuse yellow pigmentation (dyp);

    M. Septate fungal hyphae; N. Monophialidic conidiogenous cells; O. Occurrence of

    microconidia in short chain; P.-R. Microconidia of different shapes- P. oval, Q. fusiform,

    R. napiform; S. Banana-shaped macroconidia; T. Experimentally diseased plants in

    pathogenicity test; U. Control plants in pathogenicity test; V. Magnified view of

    experimentally diseased plants showing same disease symptoms; W. Growth pattern of

    pure cultured colony prepared from re-isolated pathogens from experimental disease

    plants showing white mycelium and diffuse yellow pigmentation

  12. Continued part 3:

    characteristics macro and micro conidia (Table 1). The control plants, however, did not

    show any disease symptoms and no traces of F. thapsinum were recovered from them.

    Thorough literature survey reveals that previously reported diseases in Azolla are

    black rots in A. pinnata from India (Kannaiyan, 1985), Rhizoctonia blight in A. japonica

    from Korea (Lee et al., 2011), and A. filiculoides from Iran (Banihashemi, 2014).

    Kanniyan and Kumar (2005) identified a fungal complex as a causal organism for black

    rot disease in A. pinnata, with Rhizoctonia solani as the primary infecting organism.

    CONCLUSION

    The present investigation reports a new record of brown rot disease in Azolla microphylla

    caused by the fungal pathogen Fusarium thapsinum. Because of the immense value of

    this plant, it will be essential to explore preventive measure(s) to protect this important

    bio-resource from destructive fungal attack.

    ACKNOWLEDGMENTS

    The financial grant is aided by UGC-MRP (F. No. 41-489/2012 (SR) dated 16th July,

    2012) and DST-PURSE programme of University of Kalyani. The first author

    acknowledges the financial support received from University of Kalyani as a University

    Research Scholar. The authors gratefully acknowledge the kind help provided by Prof.

    Animesh Kumar Datta of Botany Department, University of Kalyani. The authors

    gratefully acknowledge the valuable comments and suggestions given by anonymous

    reviewers which helped a lot to improve quality of the manuscript

  13. The water in well already has needed minerals in it so there is no need for dung or dirt just replacing water your problem is algae growing on the dirt, dung, and Azolla over nitrating the water

  14. BRIAN tray this ——–put recipient plastic ……50% soil mixed with compost 50% ……20% of recipient soil and put water and one spoon small chimics for experiement evolution

  15. problem are soil …..bottle plastic cut in long half for recipient experiment …..azzola put 10% of space water for see multiplication ,after test pass make similar into farm azzola

  16. …and hopefully the bleach will not kill your tilapia. I’m a plus 50 yr fish keeper and past fish breeder and know that the tiniest amount of bleach will kill an entire tank of its inhabitants.

  17. Brian you don’t think it could be something to do with the corn. Something in the corn falling into the pits. I don’t know just trying to help out somehow to get you back on track

  18. Perhaps too much water in the pits. If water is too deep, azolla wont get any nutrient from the bottom soil/dung. Keep water depth 4-5 inches then see what happens. Also you can contact “Azolla sa Pinas” on FB, she is azolla expert in the Philippines.

  19. A bit confused on the theory of possible diseased azolla being transferred with the scoop, seems to be the pits are connected to each other by those transfer pipes so water is able to go from one pit to another through those pipes, if the disease is on the azolla it is also in the water that the azolla lives in so will go from pit to pit via those pipes, disinfecting that scoop would be fruitless.

  20. Since all the azolla pits are linked together by overflow pipes, any disease could also flow to all the pits through the water. As you redo each pit, I would instead make it drain directly into the drainage canal beside the road to the plateau. That way each pit is isolated from the others, making it harder for a disease to spread.

  21. I think if I was you as soon as you can I would walk the soil sample through each department it has to go to and find out exactly what is going on with that property. Your pigs and chickens get sick and your crops do good and then die off, You even had fish to die off for some reason. Maybe your ground is giving off methane in large spurts or something is definitely not right.

  22. Notice lot of dead corn, whilst i appreciate the pit renewal program, makes replanting for shading, not a solution at the moment.
    Heat could be a factor?
    Have , up here in Pangasinan, been getting hotter than normal temperatures, had heat index of 111 the other day, were advising people to avoid strenuous activity outside ect.
    Could you measure the water Temperatures in the various pits.
    When too high, affects the oxygen uptake of the azolla.
    Good idea to practice hygene, wont hurt to do that.
    Be interesteing to see how the municiple azolla is doing dont think they are using the flow thru system tho? .
    But then they dont have the number of pits you do, much easier to manage the watering.

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